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  1. Alpha-1 Antitrypsin Deficiency

    MedlinePlus

    ... Liver Disease Information > Alpha-1 Antitrypsin Deficiency Alpha-1 Antitrypsin Deficiency Explore this section to learn more about alpha-1 antitrypsin deficiency, including a description of the disorder ...

  2. Alpha-1 Antitrypsin Deficiency

    MedlinePlus

    ... from the NHLBI on Twitter. What Is Alpha-1 Antitrypsin Deficiency? Alpha-1 antitrypsin (an-tee-TRIP-sin) deficiency, or AAT ... as it relates to lung disease. Overview Alpha-1 antitrypsin, also called AAT, is a protein made ...

  3. Alpha-1 Antitrypsin Test

    MedlinePlus

    ... measures the level of the protein AAT in blood. Alpha-1 antitrypsin phenotype testing evaluates the amount and type of AAT being produced and compares it to normal patterns. Alpha-1 antitrypsin genotype testing ( DNA testing) can ...

  4. Alpha-1 antitrypsin test

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/003715.htm Alpha-1 antitrypsin test To use the sharing features on this page, please enable JavaScript. Alpha-1 antitrypsin is a laboratory test to measure the ...

  5. Detecting Alpha-1 Antitrypsin Deficiency.

    PubMed

    Stoller, James K

    2016-08-01

    Alpha-1 antitrypsin deficiency is a widely underrecognized condition, with evidence of persisting long diagnostic delays and patients' frequent need to see multiple physicians before initial diagnosis. Reasons for underrecognition include inadequate understanding of alpha-1 antitrypsin deficiency by physicians and allied health care providers; failure to implement available, guideline-based practice recommendations; and the belief that effective therapy is unavailable. Multiple studies have described both the results of screening and targeted detection of individuals with alpha-1 antitrypsin deficiency, with both varying strategies employed to identify at-risk individuals and varying results of testing. Also, various strategies to enhance detection of affected individuals have been examined, including use of the electronic medical record to prompt testing and empowerment of allied health providers, especially respiratory therapists, to promote testing for alpha-1 antitrypsin deficiency. Such efforts are likely to enhance detection with the expected result that the harmful effects of delayed diagnosis can be mitigated. PMID:27564667

  6. What Causes Alpha-1 Antitrypsin Deficiency?

    MedlinePlus

    ... from the NHLBI on Twitter. What Causes Alpha-1 Antitrypsin Deficiency? Alpha-1 antitrypsin (AAT) deficiency is an inherited disease. "Inherited" ... have AAT deficiency inherit two faulty AAT genes, one from each parent. These genes tell cells in ...

  7. How Is Alpha-1 Antitrypsin Deficiency Treated?

    MedlinePlus

    ... from the NHLBI on Twitter. How Is Alpha-1 Antitrypsin Deficiency Treated? Alpha-1 antitrypsin (AAT) deficiency has no cure, but its ... of these treatments are the same as the ones used for a lung disease called COPD (chronic ...

  8. Genetics Home Reference: alpha-1 antitrypsin deficiency

    MedlinePlus

    ... and genetic modifiers of emphysema risk. Thorax. 2004 Mar;59(3):259-64. Review. Citation on PubMed ... alpha}1-antitrypsin deficiency. Arch Intern Med. 2009 Mar 23;169(6):546-50. doi: 10.1001/ ...

  9. Alpha-1 Antitrypsin Deficiency (Inherited Emphysema)

    MedlinePlus

    ... 1 protein in the blood with normal alpha-1 antitrypsin from healthy plasma donors. It is given in a vein (IV). The dose is adjusted based on body weight. This treatment is often given once a week. There are three ... the management of Alpha-1 related emphysema includes: • Exercise and a healthy lifestyle ...

  10. [Alpha-1 antitrypsin deficiency: diagnosis and treatment].

    PubMed

    Camelier, Aquiles A; Winter, Daniel Hugo; Jardim, José Roberto; Barboza, Carlos Eduardo Galvão; Cukier, Alberto; Miravitlles, Marc

    2008-07-01

    Alpha-1 antitrypsin deficiency is a recently identified genetic disease that occurs almost as frequently as cystic fibrosis. It is caused by various mutations in the SERPINA1 gene, and has numerous clinical implications. Alpha-1 antitrypsin is mainly produced in the liver and acts as an antiprotease. Its principal function is to inactivate neutrophil elastase, preventing tissue damage. The mutation most commonly associated with the clinical disease is the Z allele, which causes polymerization and accumulation within hepatocytes. The accumulation of and the consequent reduction in the serum levels of alpha-1 antitrypsin cause, respectively, liver and lung disease, the latter occurring mainly as early emphysema, predominantly in the lung bases. Diagnosis involves detection of low serum levels of alpha-1 antitrypsin as well as phenotypic confirmation. In addition to the standard treatment of chronic obstructive pulmonary disease, specific therapy consisting of infusion of purified alpha-1 antitrypsin is currently available. The clinical efficacy of this therapy, which appears to be safe, has yet to be definitively established, and its cost-effectiveness is also a controversial issue that is rarely addressed. Despite its importance, in Brazil, there are no epidemiological data on the prevalence of the disease or the frequency of occurrence of deficiency alleles. Underdiagnosis has also been a significant limitation to the study of the disease as well as to appropriate treatment of patients. It is hoped that the creation of the Alpha One International Registry will resolve these and other important issues. PMID:18695797

  11. Who Is at Risk for Alpha-1 Antitrypsin Deficiency?

    MedlinePlus

    ... on Twitter. Who Is at Risk for Alpha-1 Antitrypsin Deficiency? Alpha-1 antitrypsin (AAT) deficiency occurs in all ethnic groups. ... it doesn't mean that you'll develop one of the diseases related to the condition. Some ...

  12. Delivery of Alpha-1 Antitrypsin to Airways.

    PubMed

    Griese, Matthias; Scheuch, Gerhard

    2016-08-01

    Treatment with exogenous alpha-1 antitrypsin (AAT), a potent serine protease inhibitor, was developed originally for chronic obstructive pulmonary disease associated with AAT deficiency; however, other lung conditions involving neutrophilic inflammation and proteolytic tissue injury related to neutrophil elastase and other serine proteases may also be considered for AAT therapy. These conditions include bronchiectasis caused by primary ciliary dyskinesia, cystic fibrosis, and other diseases associated with an increased free elastase activity in the airways. Inhaled AAT may be a viable option to counteract proteolytic tissue damage. This form of treatment requires efficient drug delivery to the targeted pulmonary compartment. Aerosol technology meeting this requirement is currently available and offers an alternative therapeutic approach to systemic AAT administration. To date, early studies in humans have shown biochemical efficacy and have established the safety of inhaled AAT. However, to bring aerosol AAT therapy to patients, large phase 3 protocols in carefully selected patient populations (i.e., subgroups of patients with AAT deficiency, cystic fibrosis, or other lung diseases with bronchiectasis) will be needed with clinical end points in addition to the measurement of proteolytic activity in the airway. The outcomes likely will have to include lung function, lung structure assessed by computed tomography imaging, disease exacerbations, health status, and mortality. PMID:27564672

  13. 21 CFR 866.5130 - Alpha-1-antitrypsin immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... the alpha-1-antitrypsin (a plasma protein) in serum, other body fluids, and tissues. The measurements... addition, alpha-1-antitrypsin deficiency has been associated with pulmonary emphysema. (b)...

  14. The Role of Neutrophils in Alpha-1 Antitrypsin Deficiency.

    PubMed

    McCarthy, Cormac; Reeves, Emer P; McElvaney, Noel G

    2016-08-01

    Alpha-1 antitrypsin deficiency (AATD) is characterized by low levels of circulating alpha-1 antitrypsin and an increased risk for emphysema, liver disease, and panniculitis. The reduced levels of alpha-1 antitrypsin in AATD predispose the lung to unopposed proteolytic activity, predominantly from neutrophil-derived proteases, chiefly neutrophil elastase. This leads to emphysema. The mechanisms subtending the liver disease are less well understood, but are probably due to a "gain-of function" inflammatory process in the liver, stoked by intracellular retention of aberrantly folded alpha-1 antitrypsin. The panniculitis associated with AATD is most likely due to unopposed proteolytic activity in the skin. Although AATD has been traditionally viewed as a condition arising from a protease-antiprotease imbalance in the lung, it is increasingly recognized that AATD is an inflammatory disorder, both in the lung and in the extrapulmonary manifestations associated with the condition. This inflammation is predominantly neutrophil driven, and there are several alpha-1 antitrypsin-related mechanisms involved in potentiating this neutrophilic response. The rationale for AAT augmentation therapy in AATD is classically based on restoring the antiprotease balance in the lung, but its beneficial effects may also be exerted systemically, further exposing the pathogenesis of AATD-related disease and indicating a potential usage for alpha-1 antitrypsin in other inflammatory conditions. PMID:27564664

  15. Deficiency Mutations of Alpha-1 Antitrypsin. Effects on Folding, Function, and Polymerization

    PubMed Central

    Haq, Imran; Saleh, Aarash D.; Dron, Louis; Regan-Mochrie, Gemma L.; Motamedi-Shad, Neda; Hurst, John R.; Gooptu, Bibek

    2016-01-01

    Misfolding, polymerization, and defective secretion of functional alpha-1 antitrypsin underlies the predisposition to severe liver and lung disease in alpha-1 antitrypsin deficiency. We have identified a novel (Ala336Pro, Baghdad) deficiency variant and characterized it relative to the wild-type (M) and Glu342Lys (Z) alleles. The index case is a homozygous individual of consanguineous parentage, with levels of circulating alpha-1 antitrypsin in the moderate deficiency range, but is a biochemical phenotype that could not be classified by standard methods. The majority of the protein was present as functionally inactive polymer, and the remaining monomer was 37% active relative to the wild-type protein. These factors combined indicate an 85 to 95% functional deficiency, similar to that seen with ZZ homozygotes. Biochemical, biophysical, and computational studies further defined the molecular basis of this deficiency. These studies demonstrated that native Ala336Pro alpha-1 antitrypsin could populate the polymerogenic intermediate—and therefore polymerize—more readily than either wild-type alpha-1 antitrypsin or the Z variant. In contrast, folding was far less impaired in Ala336Pro alpha-1 antitrypsin than in the Z variant. The data are consistent with a disparate contribution by the “breach” region and “shutter” region of strand 5A to folding and polymerization mechanisms. Moreover, the findings demonstrate that, in these variants, folding efficiency does not correlate directly with the tendency to polymerize in vitro or in vivo. They therefore differentiate generalized misfolding from polymerization tendencies in missense variants of alpha-1 antitrypsin. Clinically, they further support the need to quantify loss-of-function in alpha-1 antitrypsin deficiency to individualize patient care. PMID:26091018

  16. Deficiency Mutations of Alpha-1 Antitrypsin. Effects on Folding, Function, and Polymerization.

    PubMed

    Haq, Imran; Irving, James A; Saleh, Aarash D; Dron, Louis; Regan-Mochrie, Gemma L; Motamedi-Shad, Neda; Hurst, John R; Gooptu, Bibek; Lomas, David A

    2016-01-01

    Misfolding, polymerization, and defective secretion of functional alpha-1 antitrypsin underlies the predisposition to severe liver and lung disease in alpha-1 antitrypsin deficiency. We have identified a novel (Ala336Pro, Baghdad) deficiency variant and characterized it relative to the wild-type (M) and Glu342Lys (Z) alleles. The index case is a homozygous individual of consanguineous parentage, with levels of circulating alpha-1 antitrypsin in the moderate deficiency range, but is a biochemical phenotype that could not be classified by standard methods. The majority of the protein was present as functionally inactive polymer, and the remaining monomer was 37% active relative to the wild-type protein. These factors combined indicate an 85 to 95% functional deficiency, similar to that seen with ZZ homozygotes. Biochemical, biophysical, and computational studies further defined the molecular basis of this deficiency. These studies demonstrated that native Ala336Pro alpha-1 antitrypsin could populate the polymerogenic intermediate-and therefore polymerize-more readily than either wild-type alpha-1 antitrypsin or the Z variant. In contrast, folding was far less impaired in Ala336Pro alpha-1 antitrypsin than in the Z variant. The data are consistent with a disparate contribution by the "breach" region and "shutter" region of strand 5A to folding and polymerization mechanisms. Moreover, the findings demonstrate that, in these variants, folding efficiency does not correlate directly with the tendency to polymerize in vitro or in vivo. They therefore differentiate generalized misfolding from polymerization tendencies in missense variants of alpha-1 antitrypsin. Clinically, they further support the need to quantify loss-of-function in alpha-1 antitrypsin deficiency to individualize patient care.

  17. Deficiency of a alpha-1-antitrypsin influences systemic iron homeostasis

    EPA Science Inventory

    Abstract Background: There is evidence that proteases and anti-proteases participate in the iron homeostasis of cells and living systems. We tested the postulate that alpha-1 antitrypsin (A1AT) polymorphism and the consequent deficiency of this anti-protease in humans are asso...

  18. 21 CFR 866.5130 - Alpha-1-antitrypsin immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5130 Alpha-1-antitrypsin immunological test system. (a) Identification. An alpha-1-antitrypsin... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alpha-1-antitrypsin immunological test system....

  19. New Concepts in Alpha-1 Antitrypsin Deficiency Disease Mechanisms.

    PubMed

    Marciniak, Stefan J; Ordóñez, Adriana; Dickens, Jennifer A; Chambers, Joseph E; Patel, Vruti; Dominicus, Caia S; Malzer, Elke

    2016-08-01

    Alpha-1 antitrypsin deficiency is predominantly caused by point mutations that alter the protein's folding. These mutations fall into two broad categories: those that destabilize the protein dramatically and lead to its post-translational degradation and those that affect protein structure more subtly to promote protein polymerization within the endoplasmic reticulum (ER). This distinction is important because it determines the cell's response to each mutant. The severely misfolded mutants trigger an unfolded protein response (UPR) that promotes improved protein folding but can kill the cell in the chronic setting. In contrast, mutations that permit polymer formation fail to activate the UPR but instead promote a nuclear factor-κB-mediated ER overload response. The ability of polymers to increase a cell's sensitivity to ER stress likely explains apparent inconsistencies in the alpha-1 antitrypsin-signaling literature that have linked polymers with the UPR. In this review we discuss the use of mutant serpins to dissect each signaling pathway. PMID:27564663

  20. Biomarkers in Alpha-1 Antitrypsin Deficiency Chronic Obstructive Pulmonary Disease.

    PubMed

    Turino, Gerard M; Ma, Shuren; Cantor, Jerome O; Lin, Yong Y

    2016-08-01

    Biomarkers of pathogenesis in chronic obstructive pulmonary disease (COPD) can significantly accelerate drug development. In COPD related to alpha-1 antitrypsin deficiency, the role of neutrophil elastase and its inhibition by alpha-1 antitrypsin protein focused interest on elastin degradation and the development of pulmonary emphysema. Amino acids desmosine and isodesmosine are unique cross-links in mature elastin fibers and can serve as biomarkers of elastin degradation when measured in body fluids. This review gives a perspective on what has been learned by the earliest measurements of desmosine and isodesmosine followed by later studies using methods of increased sensitivity and specificity and the meaning for developing new therapies. Also included are brief statements on the biomarkers fibrinogen, CC-16, and Aa-Val-360 in COPD. PMID:27564670

  1. Alpha-1 Antitrypsin Deficiency: Beyond the Protease/Antiprotease Paradigm.

    PubMed

    Cosio, Manuel G; Bazzan, Erica; Rigobello, Chiara; Tinè, Mariaenrica; Turato, Graziella; Baraldo, Simonetta; Saetta, Marina

    2016-08-01

    From the discovery that alpha-1 antitrypsin (AAT) was an effective inhibitor of neutrophil elastase originated the classic paradigm of protease/antiprotease imbalance, linking lung destruction to the unopposed effect of proteases in patients with the deficiency. Notwithstanding its importance as an antiprotease, it has become evident that alpha-1 antitrypsin has important antiinflammatory and immune-regulatory activities, which may be critically involved in lung destruction. We review here recent evidence showing that, indeed, an important adaptive immune reaction is present in lungs with AAT deficiency, similar to the one seen in severe chronic obstructive pulmonary disease with normal AAT. On the basis of recent evidence from epidemiological, clinical, and pathogenetic studies, it is likely time to move on from the original protease/antiprotease hypothesis for the production of emphysema toward a more complex paradigm, involving the antiinflammatory and immune modulating functions of AAT. PMID:27564665

  2. Learning about Alpha-1 Antitrypsin Deficiency (AATD)

    MedlinePlus

    ... Links for Patient Care Education All About the Human Genome Project Fact Sheets Genetic Education Resources for Teachers Genomic Careers National DNA Day Online Education Kit Online Genetics Education ... Subjects Research Informed Consent for Genomics Research Intellectual ...

  3. Randomized, Placebo-Controlled Trials in Alpha-1 Antitrypsin Deficiency.

    PubMed

    Sandhaus, Robert A

    2016-08-01

    Alpha-1 antitrypsin deficiency (AATD) is a condition caused by the inheritance of two mutated SERPINA1 gene alleles. Individuals with AATD are at increased risk of injury to the liver and lungs. The pulmonary manifestations include precocious onset of pulmonary emphysema and bronchiectasis. For nearly three decades, treatment has been available to individuals with emphysema caused by AATD, but this therapy-augmentation of plasma and tissue alpha-1 antitrypsin levels by intravenous administration of human plasma-derived protein-was approved by regulatory authorities based on its biochemical efficacy. This therapy appears to slow the progression of emphysema in patients with AATD. The medical, patient, and regulatory communities have sought assurance that this expensive therapy provides measurable clinical benefit. Documenting such benefit has been difficult because of the slow progression of the underlying lung disease in AATD, the rarity of this genetic condition, and the lack of direct quantitative measurements of emphysema progression. Over the past decade, quantitative computed tomography (CT) densitometry of the lungs has been found to correlate with severity and progression of emphysema. The recent publication of a well-powered, masked, placebo-controlled study using CT densitometry to evaluate the effectiveness of augmentation therapy at slowing the progression of emphysema has provided some assurance of the clinical efficacy of this therapy. PMID:27564674

  4. Alpha 1-antitrypsin and survival in hepatocellular carcinoma.

    PubMed Central

    Tzonou, A.; Sparos, L.; Kalapothaki, V.; Zavitsanos, X.; Rebelakos, A.; Trichopoulos, D.

    1990-01-01

    The association between serum levels of alpha 1-antitrypsin (alpha 1 AT) at the time of diagnosis and survival was studied in a group of 78 patients with confirmed hepatocellular carcinoma (HCC). All 78 patients were followed until the time of death, which occurred in all instances from HCC, with a median time of 6 months and a range of 1-117 months. Cox's proportional hazards model was utilised in the analysis controlling for sex, age, HBsAg status and logarithmically transformed values of alpha-fetoprotein (alpha-FP). Older patients and patients positive for HBsAg have suggestively higher fatality rates (0.05 less than P less than 0.10) whereas in these data sex and AFP levels were not important prognostic factors. Increased levels of serum at alpha 1AT at the time of diagnosis of HCC were statistically significantly (P less than 0.05) related with shorter survival, patients with higher serum alpha 1AT by 200 mg 100 ml-1 having an expected survival time shorter by about 25%. PMID:2153397

  5. 21 CFR 866.5130 - Alpha-1-antitrypsin immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alpha-1-antitrypsin immunological test system. 866.5130 Section 866.5130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... the alpha-1-antitrypsin (a plasma protein) in serum, other body fluids, and tissues. The...

  6. An ECLIPSE View of Alpha-1 Antitrypsin Deficiency.

    PubMed

    Lomas, David A

    2016-08-01

    Chronic obstructive pulmonary disease (COPD) is a multicomponent condition that is estimated to become the third leading cause of death in 2020. The ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints) study, funded by GlaxoSmithKline, is an observational study designed to define outcomes that can be used as endpoints in clinical trials in individuals with COPD. It allowed us to describe the heterogeneity of COPD, the stability of the exacerbation phenotype, and the factors associated with a progressive decline in lung function and the progression of emphysema on computed tomography scans. The cohort was also used to define genetic factors and biomarkers associated with COPD and disease progression. This review considers how the results from ECLIPSE can inform our understanding of the lung disease associated with alpha-1 antitrypsin deficiency. PMID:27564668

  7. Gene Therapy for Alpha-1 Antitrypsin Deficiency Lung Disease.

    PubMed

    Chiuchiolo, Maria J; Crystal, Ronald G

    2016-08-01

    Alpha-1 antitrypsin (AAT) deficiency, characterized by low plasma levels of the serine protease inhibitor AAT, is associated with emphysema secondary to insufficient protection of the lung from neutrophil proteases. Although AAT augmentation therapy with purified AAT protein is efficacious, it requires weekly to monthly intravenous infusion of AAT purified from pooled human plasma, has the risk of viral contamination and allergic reactions, and is costly. As an alternative, gene therapy offers the advantage of single administration, eliminating the burden of protein infusion, and reduced risks and costs. The focus of this review is to describe the various strategies for AAT gene therapy for the pulmonary manifestations of AAT deficiency and the state of the art in bringing AAT gene therapy to the bedside. PMID:27564673

  8. Active Trafficking of Alpha 1 Antitrypsin across the Lung Endothelium

    PubMed Central

    Lockett, Angelia D.; Brown, Mary Beth; Santos-Falcon, Nieves; Rush, Natalia I.; Oueini, Houssam; Oberle, Amber J.; Bolanis, Esther; Fragoso, Miryam A.; Petrusca, Daniela N.; Serban, Karina A.; Schweitzer, Kelly S.; Presson Jr., Robert G.

    2014-01-01

    The homeostatic lung protective effects of alpha-1 antitrypsin (A1AT) may require the transport of circulating proteinase inhibitor across an intact lung endothelial barrier. We hypothesized that uninjured pulmonary endothelial cells transport A1AT to lung epithelial cells. Purified human A1AT was rapidly taken up by confluent primary rat pulmonary endothelial cell monolayers, was secreted extracellularly, both apically and basolaterally, and was taken up by adjacent rat lung epithelial cells co-cultured on polarized transwells. Similarly, polarized primary human lung epithelial cells took up basolaterally-, but not apically-supplied A1AT, followed by apical secretion. Evidence of A1AT transcytosis across lung microcirculation was confirmed in vivo by two-photon intravital microscopy in mice. Time-lapse confocal microscopy indicated that A1AT co-localized with Golgi in the endothelium whilst inhibition of the classical secretory pathway with tunicamycin significantly increased intracellular retention of A1AT. However, inhibition of Golgi secretion promoted non-classical A1AT secretion, associated with microparticle release. Polymerized A1AT or A1AT supplied to endothelial cells exposed to soluble cigarette smoke extract had decreased transcytosis. These results suggest previously unappreciated pathways of A1AT bidirectional uptake and secretion from lung endothelial cells towards the alveolar epithelium and airspaces. A1AT trafficking may determine its functional bioavailablity in the lung, which could be impaired in individuals exposed to smoking or in those with A1AT deficiency. PMID:24743137

  9. Alpha 1 Antitrypsin Deficiency in Infants with Neonatal Cholestasis

    PubMed Central

    Monajemzadeh, Maryam; Shahsiah, Reza; Vasei, Mohammad; Tanzifi, Parin; Rezaei, Nima; Najafi, Mehri; Soleimanifar, Narjes; Eghbali, Maryam

    2013-01-01

    Objective Alpha1-antitrypsin deficiency (A1ATD) is the most important indication for liver transplantation in children. The gene frequencies vary in different ethnic groups. In the present study, we attempt to determine the frequencies of the most common defective alleles, Z and S, in Iranian children suffering from idiopathic neonatal cholestasis. Eighty-seven infants were typed for Z and S alleles. Methods In a single center study, 87 consecutive liver biopsies from infants with cholestasis were reviewed and patients with neonatal cholestasis enrolled in the study and cases with confirmed biliary tract atresia excluded. Formalin fixed paraffin embedded blocks were used for DNA extraction. AAT genotype was determined by polymerase chain reaction (PCR) assay and amplification of the two most common deficiency variants, S and Z alleles, and then sequencing of PCR products. Findings There were 48 (55.2%) males and 39 (44.8%) females, with a median age of 60 days. Out of 87 of the study subject, 2 (2.2%) were heterozygous for the S allele, and no ZZ, SS or MZ individual was found in the patients. No other polymorphism was found in the sequencing results. Conclusion In comparison to other populations, AAT deficiency seems not to be an important etiologic factor for neonatal cholestatic liver disease in Iran; however, further studies are recommended to estimate the true mutant gene frequencies. PMID:24800007

  10. Alpha-1 Antitrypsin Investigations Using Animal Models of Emphysema.

    PubMed

    Ni, Kevin; Serban, Karina A; Batra, Chanan; Petrache, Irina

    2016-08-01

    Animal models of disease help accelerate the translation of basic science discoveries to the bedside, because they permit experimental interrogation of mechanisms at relatively high throughput, while accounting for the complexity of an intact organism. From the groundbreaking observation of emphysema-like alveolar destruction after direct instillation of elastase in the lungs to the more clinically relevant model of airspace enlargement induced by chronic exposure to cigarette smoke, animal models have advanced our understanding of alpha-1 antitrypsin (AAT) function. Experimental in vivo models that, at least in part, replicate clinical human phenotypes facilitate the translation of mechanistic findings into individuals with chronic obstructive pulmonary disease and with AAT deficiency. In addition, unexpected findings of alveolar enlargement in various transgenic mice have led to novel hypotheses of emphysema development. Previous challenges in manipulating the AAT genes in mice can now be overcome with new transgenic approaches that will likely advance our understanding of functions of this essential, lung-protective serine protease inhibitor (serpin). PMID:27564666

  11. What Are the Signs and Symptoms of Alpha-1 Antitrypsin Deficiency?

    MedlinePlus

    ... from the NHLBI on Twitter. What Are the Signs and Symptoms of Alpha-1 Antitrypsin Deficiency? The ... ability to be physically active, and wheezing. These signs and symptoms most often begin between the ages ...

  12. Systemic necrotizing vasculitides in severe alpha1-antitrypsin deficiency.

    PubMed

    Mazodier, P; Elzouki, A N; Segelmark, M; Eriksson, S

    1996-08-01

    We describe the clinical presentation and outcome in a series of eight patients with systemic necrotizing vasculitis and severe alpha1-antitrypsin (AAT) deficiency followed up at three Swedish hospitals during 1968-92. We also review six other cases reported in the literature during the same period. Diagnosis of severe AAT deficiency was based on the presence of the PiZZ phenotype, or low plasma total trypsin inhibitory capacity, or a low plasma AAT concentration (10-40% of the normal mean value) and presence of the PiSZ or PiFZ phenotype. The diagnosis of systemic vasculitis was biopsy-verified in all eight patients. Pretreatment laboratory findings, treatment protocol, and outcome were reviewed in each of the 14 patients. Of the eight patients in the Swedish series, six had systemic vasculitis of the microscopic polyangiitis form, one had Wegener's granulomatosis, and another had Henoch-Schönlein purpura. In the series as a whole (n = 14), median age at diagnosis was 48 years (range 44-84), the median number of affected organs was eight, and all 14 patients had skin involvement, and either renal or joint involvement (in most cases both); 71% (10/14) had emphysema; 57% (8/14) had hepatic abnormalities (two having cirrhosis, two fibrosis, and one multiple aneurysms in hepatic arteries); one patient who presented with acute ulcerative colitis developed manifest vasculitic syndrome three years later; and 64% (9/14) died, the major cause of death being renal failure. This syndrome, characterized by multiple organ involvement and fatal outcome, has been underdiagnosed. Physicians should be alert to the presence of the PiZ AAT deficiency gene in patients with systemic vasculitis, especially when the course is progressive or when the patient also has emphysema or cirrhosis. Awareness of those features may aid prompt recognition and enable early treatment.

  13. Rationale and Design of the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis Study. Alpha-1 Protocol.

    PubMed

    Strange, Charlie; Senior, Robert M; Sciurba, Frank; O'Neal, Scott; Morris, Alison; Wisniewski, Stephen R; Bowler, Russell; Hochheiser, Harry S; Becich, Michael J; Zhang, Yingze; Leader, Joseph K; Methé, Barbara A; Kaminski, Naftali; Sandhaus, Robert A

    2015-10-01

    Severe deficiency of alpha-1 antitrypsin has a highly variable clinical presentation. The Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis α1 Study is a prospective, multicenter, cross-sectional study of adults older than age 35 years with PiZZ or PiMZ alpha-1 antitrypsin genotypes. It is designed to better understand if microbial factors influence this heterogeneity. Clinical symptoms, pulmonary function testing, computed chest tomography, exercise capacity, and bronchoalveolar lavage (BAL) will be used to define chronic obstructive pulmonary disease (COPD) phenotypes that can be studied with an integrated systems biology approach that includes plasma proteomics; mouth, BAL, and stool microbiome and virome analysis; and blood microRNA and blood mononuclear cell RNA and DNA profiling. We will rely on global genome, transcriptome, proteome, and metabolome datasets. Matched cohorts of PiZZ participants on or off alpha-1 antitrypsin augmentation therapy, PiMZ participants not on augmentation therapy, and control participants from the Subpopulations and Intermediate Outcome Measures in COPD Study who match on FEV1 and age will be compared. In the primary analysis, we will determine if the PiZZ individuals on augmentation therapy have a difference in lower respiratory tract microbes identified compared with matched PiZZ individuals who are not on augmentation therapy. By characterizing the microbiome in alpha-1 antitrypsin deficiency (AATD), we hope to define new phenotypes of COPD that explain some of the diversity of clinical presentations. As a unique genetic cause of COPD, AATD may inform typical COPD pathogenesis, and better understanding of it may illuminate the complex interplay between environment and genetics. Although the biologic approaches are hypothesis generating, the results may lead to development of novel biomarkers, better understanding of COPD phenotypes, and development of novel diagnostic and therapeutic trials in AATD and COPD

  14. Rationale and Design of the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis Study. Alpha-1 Protocol

    PubMed Central

    Senior, Robert M.; Sciurba, Frank; O’Neal, Scott; Morris, Alison; Wisniewski, Stephen R.; Bowler, Russell; Hochheiser, Harry S.; Becich, Michael J.; Zhang, Yingze; Leader, Joseph K.; Methé, Barbara A.; Kaminski, Naftali; Sandhaus, Robert A.

    2015-01-01

    Severe deficiency of alpha-1 antitrypsin has a highly variable clinical presentation. The Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis α1 Study is a prospective, multicenter, cross-sectional study of adults older than age 35 years with PiZZ or PiMZ alpha-1 antitrypsin genotypes. It is designed to better understand if microbial factors influence this heterogeneity. Clinical symptoms, pulmonary function testing, computed chest tomography, exercise capacity, and bronchoalveolar lavage (BAL) will be used to define chronic obstructive pulmonary disease (COPD) phenotypes that can be studied with an integrated systems biology approach that includes plasma proteomics; mouth, BAL, and stool microbiome and virome analysis; and blood microRNA and blood mononuclear cell RNA and DNA profiling. We will rely on global genome, transcriptome, proteome, and metabolome datasets. Matched cohorts of PiZZ participants on or off alpha-1 antitrypsin augmentation therapy, PiMZ participants not on augmentation therapy, and control participants from the Subpopulations and Intermediate Outcome Measures in COPD Study who match on FEV1 and age will be compared. In the primary analysis, we will determine if the PiZZ individuals on augmentation therapy have a difference in lower respiratory tract microbes identified compared with matched PiZZ individuals who are not on augmentation therapy. By characterizing the microbiome in alpha-1 antitrypsin deficiency (AATD), we hope to define new phenotypes of COPD that explain some of the diversity of clinical presentations. As a unique genetic cause of COPD, AATD may inform typical COPD pathogenesis, and better understanding of it may illuminate the complex interplay between environment and genetics. Although the biologic approaches are hypothesis generating, the results may lead to development of novel biomarkers, better understanding of COPD phenotypes, and development of novel diagnostic and therapeutic trials in AATD and COPD

  15. Rationale and Design of the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis Study. Alpha-1 Protocol.

    PubMed

    Strange, Charlie; Senior, Robert M; Sciurba, Frank; O'Neal, Scott; Morris, Alison; Wisniewski, Stephen R; Bowler, Russell; Hochheiser, Harry S; Becich, Michael J; Zhang, Yingze; Leader, Joseph K; Methé, Barbara A; Kaminski, Naftali; Sandhaus, Robert A

    2015-10-01

    Severe deficiency of alpha-1 antitrypsin has a highly variable clinical presentation. The Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis α1 Study is a prospective, multicenter, cross-sectional study of adults older than age 35 years with PiZZ or PiMZ alpha-1 antitrypsin genotypes. It is designed to better understand if microbial factors influence this heterogeneity. Clinical symptoms, pulmonary function testing, computed chest tomography, exercise capacity, and bronchoalveolar lavage (BAL) will be used to define chronic obstructive pulmonary disease (COPD) phenotypes that can be studied with an integrated systems biology approach that includes plasma proteomics; mouth, BAL, and stool microbiome and virome analysis; and blood microRNA and blood mononuclear cell RNA and DNA profiling. We will rely on global genome, transcriptome, proteome, and metabolome datasets. Matched cohorts of PiZZ participants on or off alpha-1 antitrypsin augmentation therapy, PiMZ participants not on augmentation therapy, and control participants from the Subpopulations and Intermediate Outcome Measures in COPD Study who match on FEV1 and age will be compared. In the primary analysis, we will determine if the PiZZ individuals on augmentation therapy have a difference in lower respiratory tract microbes identified compared with matched PiZZ individuals who are not on augmentation therapy. By characterizing the microbiome in alpha-1 antitrypsin deficiency (AATD), we hope to define new phenotypes of COPD that explain some of the diversity of clinical presentations. As a unique genetic cause of COPD, AATD may inform typical COPD pathogenesis, and better understanding of it may illuminate the complex interplay between environment and genetics. Although the biologic approaches are hypothesis generating, the results may lead to development of novel biomarkers, better understanding of COPD phenotypes, and development of novel diagnostic and therapeutic trials in AATD and COPD

  16. Alpha 1-Antitrypsin Therapy Mitigated Ischemic Stroke Damage in Rats

    PubMed Central

    Moldthan, Huong L.; Hirko, Aaron C.; Thinschmidt, Jeffrey S.; Grant, Maria; Li, Zhimin; Peris, Joanna; Lu, Yuanqing; Elshikha, Ahmed; King, Michael A.; Hughes, Jeffrey A.; Song, Sihong

    2014-01-01

    Currently, the only effective therapy for acute ischemic stroke is the thrombolytic agent recombinant tissue plasminogen activator. α1-Antitrypsin, an endogenous inhibitor of serine proteinases and a primary acute phase protein with potent anti-inflammatory, anti-apoptotic, antimicrobial and cytoprotective activities, could be beneficial in stroke.. The goal of this study was to test whether α1-antitrypsin could improve ischemic stroke outcome in an established rat model. Middle cerebral artery occlusion was induced in male rats via intracranial microinjection of endothelin-1. Five to ten minutes following stroke induction rats received either intracranial or intravenous delivery of human α1-antitrypsin. Cylinder and vibrissae tests were used to evaluate sensorimotor function before and 72 hours after middle cerebral artery occlusion. Infarct volumes were examined via either 2,3,5-triphenyltetrazolium chloride assay or magnetic resonance imaging 72 hours after middle cerebral artery occlusion. Despite equivalent initial strokes, at 72 hours the infarct volumes of the human α1-antitrypsin treatment groups (local and systemic injection) were statistically significantly reduced by 83% and 63% (p<0.0001 and p < 0.05 respectively) compared with control rats. Human α1-antitrypsin significantly limited sensory motor systems deficits. Human α1-antitrypsin could be a potential novel therapeutic drug for the protection against neurodegeneration following ischemic stroke, but more studies are needed to investigate the protective mechanisms and efficacy in other animal models. PMID:24582784

  17. Immunohistochemical demonstration of alpha 1-antichymotrypsin and alpha 1-antitrypsin in salivary gland pleomorphic adenomas of children.

    PubMed

    Takahashi, H; Fujita, S; Tsuda, N; Tezuka, F; Okabe, H

    1990-09-01

    Twenty-five benign pleomorphic adenomas of salivary glands in children were studied with immunohistochemical techniques in order to characterize the cell types comprising the epithelial and so-called "mesenchymal" regions of the tumors. The antisera against alpha 1-antichymotrypsin (alpha 1-ACT) and alpha 1-antitrypsin (alpha 1-AT) were used to stain in normal salivary gland tissue as well as in pleomorphic adenoma. In normal salivary glands, alpha 1-ACT was localized to the intercalated duct and serous acinar cells. On the other hand, there was positive staining for alpha 1-AT in the intercalated and striated duct cells. Twenty-five cases (100%) of pleomorphic adenomas in children displayed positivity to alpha 1-ACT staining and 22 cases (88%) showed a positive staining for alpha 1-AT. alpha 1-ACT staining was particularly intense in chondrocyte-like cells of 20 cases (80%), in inner tubular cells of 16 (64%) and cyst-lining cells of 12 (52%). The limited number of tumor cells which were called plasmatoid or hyaline cells and squamous epithelial cells, were positive for alpha 1-ACT. None of the outer tubular cells and hyalinous material was positively stained for alpha 1-ACT. A strong positive reaction for alpha 1-AT was observed in chondrocyte-like cells of 15 cases (60%). Inner tubular cells were positive for alpha 1-AT in 12 cases (48%), plasmatoid or hyaline cells in 10 (40%) and cyst-lining cells in 8 (35%). Squamous epithelial cells, clear cells, secretory product and hyalinous material were positive for alpha 1-AT in some cases. Chondroid matrix and myxoid stroma had no reaction with both antibodies. The biological role of alpha 1-ACT and alpha 1-AT with a wide immunohistochemical distribution in pleomorphic adenomas of children may be associated with a self regulating mechanism which inhibits degradation by tissue proteinases.

  18. Trials and Tribulations: An Industry Perspective on Conducting Registrational Trials in Alpha-1 Antitrypsin Deficiency.

    PubMed

    Forshag, Mark S

    2016-08-01

    Registrational trials in rare and orphan diseases present complexities related to the identification of subjects, recruitment, logistical hurdles incumbent with far-flung study sites, and end points that are often less well defined than are those used in more common illnesses. Alpha-1 antitrypsin deficiency is an orphan disease of genetic origin that carries the additional challenges of variable penetration and slow disease progression. Registrational trials of augmentation therapy using plasma-derived alpha-1 antitrypsin carry all of the above-noted burdens, as well as competition from commercially available augmentation therapy in many countries. PMID:27564675

  19. Diagnosis of alpha-1 antitrypsin deficiency: a population-based study

    PubMed Central

    Barrecheguren, Miriam; Monteagudo, Mónica; Simonet, Pere; Llor, Carl; Rodriguez, Esther; Ferrer, Jaume; Esquinas, Cristina; Miravitlles, Marc

    2016-01-01

    Introduction Alpha-1 antitrypsin deficiency (AATD) remains an underdiagnosed condition despite initiatives developed to increase awareness. The objective was to describe the current situation of the diagnosis of AATD in primary care (PC) in Catalonia, Spain. Methods We performed a population-based study with data from the Information System for Development in Research in Primary Care, a population database that contains information of 5.8 million inhabitants (80% of the population of Catalonia). We collected the number of alpha-1 antitrypsin (AAT) determinations performed in the PC in two periods (2007–2008 and 2010–2011) and described the characteristics of the individuals tested. Results A total of 12,409 AAT determinations were performed (5,559 in 2007–2008 and 6,850 in 2010–2011), with 10.7% of them in children. As a possible indication for AAT determination, 28.9% adults and 29.4% children had a previous diagnosis of a disease related to AATD; transaminase levels were above normal in 17.7% of children and 47.1% of adults. In total, 663 (5.3%) individuals had intermediate AATD (50–100 mg/dL), 24 (0.2%) individuals had a severe deficiency (<50 mg/dL), with a prevalence of 0.19 cases of severe deficiency per 100 determinations. Nine (41%) of the adults with severe deficiency had a previous diagnosis of COPD/emphysema, and four (16.7%) were diagnosed with COPD within 6 months. Conclusion The number of AAT determinations in the PC is low in relation to the prevalence of COPD but increased slightly along the study period. The indication to perform the test is not always clear, and patients detected with deficiency are not always referred to a specialist. PMID:27274221

  20. Alpha-1 antitrypsin reduces ovariectomy-induced bone loss in mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alpha-1antitrypsin (AAT) is a multifunctional protein with proteinase inhibitor and anti-inflammatory activities. Recent studies showed that AAT has therapeutic effect for diseases associated with inflammation, such as type 1 diabetes and arthritis. Proinflammatory cytokines are primary mediators of...

  1. 21 CFR 866.5130 - Alpha-1-antitrypsin immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alpha-1-antitrypsin immunological test system. 866.5130 Section 866.5130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  2. 21 CFR 866.5130 - Alpha-1-antitrypsin immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alpha-1-antitrypsin immunological test system. 866.5130 Section 866.5130 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  3. Molecular characterization of the new defective P(brescia) alpha1-antitrypsin allele.

    PubMed

    Medicina, Daniela; Montani, Nadia; Fra, Anna M; Tiberio, Laura; Corda, Luciano; Miranda, Elena; Pezzini, Alessandro; Bonetti, Fausta; Ingrassia, Rosaria; Scabini, Roberta; Facchetti, Fabio; Schiaffonati, Luisa

    2009-08-01

    Alpha1-antitrypsin (alpha(1)AT) deficiency is a hereditary disorder associated with reduced alpha(1)AT serum level, predisposing adults to pulmonary emphysema. Among the known mutations of the alpha(1)AT gene (SERPINA1) causing alpha(1)AT deficiency, a few alleles, particularly the Z allele, may also predispose adults to liver disease. We have characterized a new defective alpha(1)AT allele (c.745G>C) coding for a mutant alpha(1)AT (Gly225Arg), named P(brescia). The P(brescia) alpha(1)AT allele was first identified in combination with the rare defective M(würzburg) allele in an 11-year-old boy showing significantly reduced serum alpha(1)AT level. Subsequently, the P(brescia) allele was found in the heterozygous state with the normal M or the defective Z allele in nine and three adults respectively. In cellular models of the disease, we show that the P(brescia) mutant is retained in the endoplasmic reticulum as ordered polymers and is secreted more slowly than the normal M alpha(1)AT. This behaviour recapitulates the abnormal cellular handling and fate of the Z alpha(1)AT and suggests that the mutation present in the P(brescia) alpha(1)AT causes a conformational change of the protein which, by favouring polymer formation, is etiologic to both severe alpha(1)AT deficiency in the plasma and toxic protein-overload in the liver.

  4. Analysis of the endoplasmic reticular Ca2+ requirement for alpha1-antitrypsin processing and transport competence.

    PubMed Central

    Cooper, G R; Brostrom, C O; Brostrom, M A

    1997-01-01

    Depletion of Ca2+ sequestered within the endoplasmic reticulum (ER) of HepG2 hepatoma cells results in the luminal accumulation of immature alpha1-antitrypsin possessing Man8-9 GlcNAc2 oligosaccharide side chains. This study explores the basis for this arrest and describes consequent alterations in the size and rate of secretion of the complex endoglycosidase H-resistant form of the protein. Inhibition of glucosidase I and II with castanospermine or alpha-1,2-mannosidase with 1-deoxymannojirimycin produced altered ER processing intermediates that were rapidly secreted. Subsequent mobilization of ER Ca2+ stores resulted in the appearance and retention of slightly larger related forms of these intermediates. Retention of glycosylated intermediates was not ascribable to an association with alpha1,2-mannosidase or lectin-like chaperones, the intermediates were not degraded and all evidence of ER retention or size alterations produced by Ca2+ depletion was quickly reversed by Ca2+ restoration. Cells that were Ca2+ depleted for 2 h slowly secreted an abnormal slightly smaller complex oligosaccharide form of alpha1-antitrypsin at approximately the same rate as the non-glycosylated protein generated by treatment with tunicamycin. The hypothesis that Ca2+ affects the folding and ER transport competence of glycosylated forms of alpha1-antitrypsin is discussed. PMID:9271078

  5. Alpha-1-antitrypsin deficiency. High prevalence in the St. Louis area determined by direct population screening.

    PubMed

    Silverman, E K; Miletich, J P; Pierce, J A; Sherman, L A; Endicott, S K; Broze, G J; Campbell, E J

    1989-10-01

    Considerable attention has been focused upon alpha-1-antitrypsin deficiency because of the insights into the pathogenesis of human pulmonary emphysema that may derive from study of deficient subjects, and because of evolving therapeutic strategies that may slow the progression of lung disease in affected persons. We have applied an automated immunoassay for alpha-1-antitrypsin to plasma samples from 20,000 blood donors. Seven PI Z antitrypsin-deficient persons were identified and confirmed; this is more than twice the number predicted from previous estimates of the Z allele frequency in the St. Louis area. Five of the subjects were further evaluated. We anticipate that this assay, if utilized to screen large populations, could identify many alpha-1-antitrypsin-deficient persons for study of the natural history of lung and liver disease associated with the deficiency. These subjects would be potential candidates for early intervention strategies to prevent the development of lung disease. The surprisingly high prevalence of deficient persons indicates that direct screening is the best method for prevalence estimation of genetic disorders.

  6. alpha-1-antitrypsin in breast milk of healthy Nigerian mothers.

    PubMed

    Omeme, J A; Lantos, J D; Ihongbe, J C

    1981-01-01

    Alpha-1-antitryspin (x-1-AT) may play a possible role as effector of immunological stasis. This study examines the levels of this glycoprotein in 73 breast milk samples from 60 healthy Nigerian mothers. Levels of x-1-AT were measured by single radial immunodiffusion according to the method of Mancini. Serum protein was measured by Lowry's method, albumin by Doumas' method. Highest mean levels of x-1-AT were found in colostrum (25 mg/dl). The level was significantly higher compared to transitional milk (14.2 mg/dl) or mature milk (165 mg/dl) (p0.001). Breast milk contains substantial amounts of x-1-AT which is not destroyed by pasturization at 56 degrees Centigrade. The immunological protective properties of breast milk are ideal for newborn babies, particularly those who are low birthweight and are thus most susceptible to neonatal necrotizing enterocolitis.

  7. Postirradiation malignant fibrous histiocytoma arising in juvenile nasopharyngeal angiofibroma and producing alpha-1-antitrypsin.

    PubMed

    Spagnolo, D V; Papadimitriou, J M; Archer, M

    1984-03-01

    A fatal nasopharyngeal malignant fibrous histiocytoma developed in a young male after irradiation of juvenile nasopharyngeal angiofibroma diagnosed 5 years earlier. The sarcoma extended from the nasopharynx into the floor of the pituitary fossa and into both parasellar regions. There was no clinical evidence of any distant spread. Many of the malignant cells contained cytoplasmic granular and globular PAS-positive inclusions shown to be alpha-1-antitrypsin immunohistochemically. Ultrastructurally, this probably corresponded to electron-dense material with distinctive patterns and which had accumulated within distended ergastoplasmic cisternae of the neoplastic cells. Three previously reported case of postirradiation sarcomas arising in nasopharyngeal angiofibroma were said to be fibrosarcomas and none produced alpha-1-antitrypsin.

  8. C-Terminal Alpha-1 Antitrypsin Peptide: A New Sepsis Biomarker with Immunomodulatory Function.

    PubMed

    Blaurock, Nancy; Schmerler, Diana; Hünniger, Kerstin; Kurzai, Oliver; Ludewig, Katrin; Baier, Michael; Brunkhorst, Frank Martin; Imhof, Diana; Kiehntopf, Michael

    2016-01-01

    Systemic inflammatory response syndrome (SIRS) is a life threatening condition and the leading cause of death in intensive care units. Although single aspects of pathophysiology have been described in detail, numerous unknown mediators contribute to the progression of this complex disease. The aim of this study was to elucidate the pathophysiological role of CAAP48, a C-terminal alpha-1 antitrypsin fragment, that we found to be elevated in septic patients and to apply this peptide as diagnostic marker for infectious and noninfectious etiologies of SIRS. Incubation of human polymorphonuclear neutrophils with synthetic CAAP48, the SNP-variant CAAP47, and several control peptides revealed intense neutrophil activation, induction of neutrophil chemotaxis, reduction of neutrophil viability, and release of cytokines. We determined the abundance of CAAP48 in patients with severe sepsis, severe SIRS of noninfectious origin, and viral infection. CAAP48 levels were 3-4-fold higher in patients with sepsis compared to SIRS of noninfectious origin and allowed discrimination of those patients with high sensitivity and specificity. Our results suggest that CAAP48 is a promising discriminatory sepsis biomarker with immunomodulatory functions, particularly on human neutrophils, supporting its important role in the host response and pathophysiology of sepsis. PMID:27382189

  9. Oxidative stress contributes to liver damage in a murine model of alpha-1-antitrypsin deficiency

    PubMed Central

    Marcus, Nancy Y; Blomenkamp, Keith; Ahmad, Muneeb; Teckman, Jeffrey H

    2012-01-01

    Alpha-1-antitrypsin deficiency is a genetic disorder, resulting in the expression of misfolded mutant protein that can polymerize and accumulate in hepatocytes, leading to liver disease in some individuals. Transgenic PiZ mice are a well characterized model, which express human alpha-1-antitrypsin mutant Z protein (ATZ protein) and faithfully recapitulate the human liver disease. Liver tissue expressing ATZ protein exhibits inflammation, injury and replacement of damaged cells. Fibrosis and hepatocellular carcinoma (HCC) develop in aging PiZ mice. In this study, microarray analysis was performed comparing young PiZ (ZY) mice to wild-type (WY) and indicated that there were alterations in gene expression levels that could influence a number of pathways leading to liver disease. Redox-regulating genes were up-regulated in ZY tissue, including carbonyl reductase 3, (CBR3), glutathione S transferase alpha 1+2, (GSTA (1+2)) and glutathione S transferase Mu 3 (GST M3). We hypothesized that oxidative stress could develop in Z mouse liver, contributing to tissue damage and disease progression with age. The results of biochemical analysis of PiZ mouse liver revealed that higher levels of reactive oxygen species (ROS) and a more oxidized, cellular redox state occurred in liver tissue from ZY mice than WY. ZY mice showed little evidence of oxidative cellular damage as assessed by protein carbonylation levels, malondialdehyde levels (MDA) and 8-oxo-7,8 dihydro-2′ deoxyguanosine (8oxodG) staining. Aging liver tissue from PiZ older mice (ZO) had elevated ROS, generally lower levels of antioxidant enzymes than younger mice and evidence of cellular damage. These data indicate that oxidative stress is a contributing factor in the development of liver disease in this model of alpha-1-antitrypsin deficiency. PMID:23104507

  10. Production of monoclonal antibodies against inactivated alpha 1-antitrypsin. Cross-reactivity with complexed alpha 1-antitrypsin and application in an assay to determine inactivated and complexed alpha 1-antitrypsin in biological fluids.

    PubMed

    Abbink, J J; Kamp, A M; Swaak, A J; Hack, C E

    1991-10-25

    15 different monoclonal antibodies (mcAbs) have been raised against the cleaved (inactive) form of the serpin alpha 1-antitrypsin (AT). In initial experiments these mcAbs were analysed for their ability to bind the native and the cleaved form of this inhibitor: eight of the 15 mcAbs appeared to react predominantly with cleaved AT. Additional experiments with mixtures of purified native AT, AT complexed to neutrophilic elastase and inactivated AT revealed that all mAbs that preferentially reacted with inactivated AT also bound to complexed AT. Using two of the mcAbs against inactivated AT a quantitative and sensitive sandwich-type radioimmunoassay was developed to determine levels of proteolytically inactivated AT in biological fluids. With this assay increased levels of inactivated AT were found in synovial fluid from patients with rheumatoid arthritis corresponding to about 2.4% (range 0.3-11%) of total AT. Approximately 10% of this inactivated AT appeared to consist of AT complexed to neutrophil elastase. The mcAbs described here further illustrate the structural resemblance between the complexed and cleaved forms of AT. In addition, these mcAbs appear to be useful tools for the study of AT in human disease.

  11. Progress with Recombinant Adeno-Associated Virus Vectors for Gene Therapy of Alpha-1 Antitrypsin Deficiency.

    PubMed

    Gruntman, Alisha M; Flotte, Terence R

    2015-06-01

    The pathway to a clinical gene therapy product often involves many changes of course and strategy before obtaining successful results. Here we outline the methodologies, both clinical and preclinical, that went into developing a gene therapy approach to the treatment of alpha-1 antitrypsin deficiency lung disease using muscle-targeted recombinant adeno-associated virus. From initial gene construct development in mouse models through multiple rounds of safety and biodistribution studies in rodents, rabbits, and nonhuman primates to ultimate human trials, this review seeks to provide insight into what clinical translation entails and could thereby inform the process for future investigators.

  12. Degradation of the human proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin by Bacteroides gingivalis.

    PubMed Central

    Carlsson, J; Herrmann, B F; Höfling, J F; Sundqvist, G K

    1984-01-01

    Various strains of black-pigmented Bacteroides species were grown on horse blood agar and suspended in human serum. After various times of incubation the effect of the bacteria on the serum was evaluated by polyacrylamide gel electrophoresis and "rocket" immunoelectrophoresis. The formation of trichloroacetic acid-soluble material in the suspensions and the capacity of the treated sera to inhibit the activity of trypsin were also determined. The two tested strains of Bacteroides gingivalis (W83, H185) degraded most serum proteins, including the plasma proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin. They did not, however, degrade alpha-1-antichymotrypsin. Bacteroides intermedius NCTC 9336, Bacteroides asaccharolyticus NCTC 9337, and an asaccharolytic oral strain different from B. gingivalis (BN11a-f) did not degrade the plasma proteinase inhibitors. These strains were, however, able to inactivate the capacity of serum to inhibit the activity of trypsin. Images PMID:6198282

  13. Proinflammatory cytokines and elastase-alpha-1-antitrypsin in Argentine hemorrhagic fever.

    PubMed

    Marta, R F; Montero, V S; Hack, C E; Sturk, A; Maiztegui, J I; Molinas, F C

    1999-01-01

    Argentine hemorrhagic fever (AHF) is a disease caused by Junin virus. In the acute phase, patients present hematologic and neurologic involvement with high levels of interferon-alpha and tumor necrosis factor-alpha (TNF-alpha. Nineteen patients with a confirmed diagnosis of AHF were studied: six severe, four moderate and nine mild cases. Serum levels of interleukin-6 (IL-6), IL-6 soluble receptor (IL-6sR), IL-8, IL-10, and elastase-alpha1-antitrypsin complex (E-alpha 1AT) were assayed by ELISAs. Levels of IL-6, IL-8, and IL-10 were high in nine, 12, and 13 patients, respectively, while levels of IL-6sR were high in two patients and low in one patient. Seven patients had increased levels of E-alpha1AT. Significant correlations were found between levels of both IL-8 and IL-10 with those of TNF-alpha as well as between IL-8 and E-alpha 1AT. These data demonstrate activation of pro-inflammatory and anti-inflammatory cytokine pathways, and statistical analysis showed differences among the clinical forms of illness. This study shows that IL-8 plays an essential role in neutrophil activation in AHF patients as demonstrated in other infectious diseases.

  14. Increased outer arm and core fucose residues on the N-glycans of mutated alpha-1 antitrypsin protein from alpha-1 antitrypsin deficient individuals.

    PubMed

    McCarthy, Cormac; Saldova, Radka; O'Brien, M Emmet; Bergin, David A; Carroll, Tomás P; Keenan, Joanne; Meleady, Paula; Henry, Michael; Clynes, Martin; Rudd, Pauline M; Reeves, Emer P; McElvaney, Noel G

    2014-02-01

    Alpha-1 antitrypsin (AAT) is the major physiological inhibitor of a range of serine proteases, and in the lung, it maintains a protease-antiprotease balance. AAT deficiency (AATD) is an autosomal co-dominant condition with the Z mutation being the most common cause. Individuals homozygous for Z (PiZZ) have low levels of circulating mutant Z-AAT protein leading to premature emphysematous lung disease. Extensive glycoanalysis has been performed on normal AAT (M-AAT) from healthy individuals and the importance of glycosylation in affecting the immune modulatory roles of AAT is documented. However, no glycoanalysis has been carried out on Z-AAT from deficient individuals to date. In this study, we investigate whether the glycans present on Z-AAT differ to those found on M-AAT from healthy controls. Plasma AAT was purified from 10 individuals: 5 AATD donors with the PiZZ phenotype and 5 PiMM healthy controls. Glycoanalysis was performed employing N-glycan release, exoglycosidase digestion and UPLC analysis. No difference in branched glycans was identified between AATD and healthy controls. However, a significant increase in both outer arm (α1-3) (p = 0.04) and core (α1-6) fucosylated glycans (p < 0.0001) was found on Z-AAT compared to M-AAT. This study has identified increased fucosylation on N-glycans of Z-AAT indicative of ongoing inflammation in AATD individuals with implications for early therapeutic intervention.

  15. Effect of swainsonine on the processing of the asparagine-linked carbohydrate chains of alpha 1-antitrypsin in rat hepatocytes. Evidence for the formation of hybrid oligosaccharides.

    PubMed

    Gross, V; Tran-Thi, T A; Vosbeck, K; Heinrich, P C

    1983-03-25

    The biosynthesis of the proteinase inhibitor alpha 1-antitrypsin has been studied in rat hepatocyte primary cultures. Newly synthesized alpha 1-antitrypsin was found in hepatocytes as a glycoprotein of an apparent molecular weight of 49,000 carrying oligosaccharide side chains of the high mannose type. In the hepatocyte medium a secreted alpha 1-antitrypsin of an apparent molecular weight of 54,000 could be identified as a glycoprotein with carbohydrate chains of the complex type. Pulse-chase experiments revealed a precursor-product relationship for the two forms of alpha 1-antitrypsin. When the hepatocytes were treated with swainsonine, an intracellular form of alpha 1-antitrypsin with an apparent molecular weight of 49,000 indistinguishable from that of control cells was found. However, the alpha 1-antitrypsin secreted from swainsonine-treated hepatocytes was different from that present in control media. It was characterized by a lower apparent molecular weight (51,000), a higher amount of [3H]mannose incorporation, half as much incorporation of [3H]galactose, and the same amount of [3H]fucose incorporation compared to alpha 1-antitrypsin of control media. In contrast to the 54,000 complex type alpha 1-antitrypsin from control media the 51,000 alpha 1-antitrypsin from the medium of swainsonine-treated cells was found to be susceptible to the action of endoglucosaminidase H, even when fucose was attached to the proximal GlcNAc residue. alpha 1-Antitrypsin secreted from swainsonine-treated cells combines features usually associated with either high mannose or complex type oligosaccharides and therefore represents a hybrid structure. In spite of its effect on the carbohydrate part of alpha 1-antitrypsin swainsonine did not impair the secretion of the incompletely processed glycoprotein. PMID:6403522

  16. Plasma elastase alpha 1-antitrypsin and lactoferrin in sepsis: evidence for neutrophils as mediators in fatal sepsis.

    PubMed

    Nuijens, J H; Abbink, J J; Wachtfogel, Y T; Colman, R W; Eerenberg, A J; Dors, D; Kamp, A J; Strack van Schijndel, R J; Thijs, L G; Hack, C E

    1992-02-01

    Increased vasopermeability and vasodilation, presumably the result of endothelial perturbation, are considered among the basic pathogenetic mechanisms in septic shock. Neutrophils have been implicated as a source for mediators in endothelial injury. We measured elastase-alpha 1-antitrypsin (alpha 1AT) complexes and lactoferrin as markers for release of neutrophil granule contents in plasma from patients with sepsis on admission to the Intensive Care Unit, and we delineated the relationship of neutrophil activation to other inflammatory parameters and to hemodynamic and biochemical parameters. Levels of elastase-alpha 1AT and lactoferrin significantly correlated with each other (r = 0.58; p less than 0.008), and were increased (greater than 3.33 and 5 nmol/L, respectively) in 96% and 71% of the patients, respectively. Lactoferrin, but not elastase-alpha 1AT, correlated with the number of white blood cells (r = 0.38; p = 0.008). Elastase-alpha 1 AT levels were significantly higher (p = 0.008), whereas white blood cell counts were lower (p = 0.015) in patients with shock when compared with patients without abnormal blood pressure. Both elastase-alpha 1AT and lactoferrin levels correlated with lactate levels (r = 0.33; p = 0.024 and r = 0.30; p = 0.04), suggesting a role for neutrophil activation in the pathogenesis of hypoxygenation. In addition, elastase-alpha 1AT correlated with the concentrations of interleukin 6 (IL-6) (r = 0.46; p = 0.001) and C3a (r = 0.38; p = 0.009), suggesting that cytokines and complement may contribute to the degranulation of neutrophils in sepsis. Elastase-alpha 1AT complexes were inversely related to C1-inhibitor (r = -0.33; p = 0.028) and to platelet numbers (r = -0.42; p = 0.003). Levels of elastase-alpha 1AT complexes in plasma appeared to be of prognostic significance; levels were higher in 27 patients who died than in 21 patients who survived (p = 0.01). The mortality in 27 patients with concentrations below 10 nM was 37%, whereas it

  17. Isolation and characterization of alpha 1-antitrypsin in PAS-positive hepatic granules from rats with experimental alpha 1-antitrypsin deficiency.

    PubMed

    Bolmer, S; Kleinerman, J

    1986-05-01

    Chronic galactosamine (GalNH2) administration in rats decreases plasma alpha 1-antitrypsin (AAT) levels to 10-50% of control levels and induces the formation of diastase-resistant, PAS-positive granules, which contain AAT in hepatocytes. This report describes the isolation and purification of hepatic granule AAT by three different methods: solubilization with guanidine hydrochloride followed by gel filtration on Bio-gel A5M, extraction with methylamine and 2-chloroethanol, and solubilization with sodium dodecyl sulfate (SDS) followed by preparative SDS-polyacrylamide gel electrophoresis. All three methods yield a single protein which precipitates with anti-rat plasma AAT antibody, and which has an apparent molecular weight of 45,000 daltons, in contrast to the molecular weight of plasma AAT, 50,000 daltons. Unlike plasma AAT, granule AAT contains no sialic acid, galactose, or fucose. Moreover, granule AAT contains a reduced amount of N-acetylglucosamine and an increased amount of mannose, compared with plasma AAT. The carbohydrate content of granule AAT varies with the isolation procedure used. Granule AAT is susceptible to cleavage by endoglucosaminidase H, which indicates the presence of high-mannose type oligosaccharides. Comparison of the molecular weight, carbohydrate composition, isoelectric point, and endoglucosaminidase H sensitivity of granule AAT isolated from rats with GalNH2-induced AAT deficiency with granule AAT from PiZ humans extends the list of similarities between experimental GalNH2-induced AAT deficiency in rats by and genetically determined AAT deficiency in humans. PMID:3085511

  18. Isolation and characterization of alpha 1-antitrypsin in PAS-positive hepatic granules from rats with experimental alpha 1-antitrypsin deficiency.

    PubMed Central

    Bolmer, S.; Kleinerman, J.

    1986-01-01

    Chronic galactosamine (GalNH2) administration in rats decreases plasma alpha 1-antitrypsin (AAT) levels to 10-50% of control levels and induces the formation of diastase-resistant, PAS-positive granules, which contain AAT in hepatocytes. This report describes the isolation and purification of hepatic granule AAT by three different methods: solubilization with guanidine hydrochloride followed by gel filtration on Bio-gel A5M, extraction with methylamine and 2-chloroethanol, and solubilization with sodium dodecyl sulfate (SDS) followed by preparative SDS-polyacrylamide gel electrophoresis. All three methods yield a single protein which precipitates with anti-rat plasma AAT antibody, and which has an apparent molecular weight of 45,000 daltons, in contrast to the molecular weight of plasma AAT, 50,000 daltons. Unlike plasma AAT, granule AAT contains no sialic acid, galactose, or fucose. Moreover, granule AAT contains a reduced amount of N-acetylglucosamine and an increased amount of mannose, compared with plasma AAT. The carbohydrate content of granule AAT varies with the isolation procedure used. Granule AAT is susceptible to cleavage by endoglucosaminidase H, which indicates the presence of high-mannose type oligosaccharides. Comparison of the molecular weight, carbohydrate composition, isoelectric point, and endoglucosaminidase H sensitivity of granule AAT isolated from rats with GalNH2-induced AAT deficiency with granule AAT from PiZ humans extends the list of similarities between experimental GalNH2-induced AAT deficiency in rats by and genetically determined AAT deficiency in humans. Images Figure 1 Figure 2 Figure 6 Figure 8 PMID:3085511

  19. Infected tracheal diverticulum: a rare association with alpha-1 antitrypsin deficiency.

    PubMed

    Amaral, Cecília Beatriz Alves; Silva, Sónia; Feijó, Salvato

    2014-01-01

    Tracheal diverticulum, defined as a benign outpouching of the tracheal wall, is rarely diagnosed in clinical practice. It can be congenital or acquired in origin, and most cases are asymptomatic, typically being diagnosed postmortem. We report a case of a 69-year-old woman who was hospitalized after presenting with fever, fatigue, pleuritic chest pain, and a right neck mass complicated by dysphagia. Her medical history was significant: pulmonary emphysema (alpha-1 antitrypsin deficiency); bronchiectasis; and thyroidectomy. On physical examination, she presented diminished breath sounds and muffled heart sounds, with a systolic murmur. Laboratory tests revealed elevated inflammatory markers, a CT scan showed an air-filled, multilocular mass in the right tracheal wall, and magnetic resonance imaging confirmed the CT findings. Fiberoptic bronchoscopy failed to reveal any abnormalities. Nevertheless, the patient was diagnosed with tracheal diverticulum. The treatment approach was conservative, consisting mainly of antibiotics. After showing clinical improvement, the patient was discharged.

  20. [Biochemical and molecular diagnosis of alpha 1 antitrypsin deficiency in a Tunisian family].

    PubMed

    Denden, S; Braham, W; Amri, F; Lakhdar, R; Lefranc, G; Knani, J; Ben Chibani, J; Haj Khelil, A

    2009-01-01

    Our study investigated alpha 1 antitrypsin deficiency (AATD) diagnosis in a family originated from central Tunisia and showing a familial history of asthma. Biochemical and genetic diagnosis for AATD was performed according to current diagnostic standards. AAT level quantification in affected individuals showed plasma AAT levels consistent with intermediate AATD (ranged from 0.91 to 1.04 g/L). The molecular analysis was assessed using the genotyping of the most prevalent PI*S and PI*Z SERPINA1 mutations and the sequencing of AAT coding exons for rare AATD variants detection. No PI*S or PI*Z deficient variants were seen in this family. Sequencing results showed the inheritance of the deficient rare variant PI*M(wurzburg) (P369S) at the heterozygous state in the mother and two affected siblings. However, AATD status remains unexplained in the third affected case, with no mutations detected in the AAT coding exons. PMID:19654085

  1. Aberrant disulphide bonding contributes to the ER retention of alpha1-antitrypsin deficiency variants.

    PubMed

    Ronzoni, Riccardo; Berardelli, Romina; Medicina, Daniela; Sitia, Roberto; Gooptu, Bibek; Fra, Anna Maria

    2016-02-15

    Mutations in alpha1-antitrypsin (AAT) can cause the protein to polymerise and be retained in the endoplasmic reticulum (ER) of hepatocytes. The ensuing systemic AAT deficiency leads to pulmonary emphysema, while intracellular polymers are toxic and cause chronic liver disease. The severity of this process varies considerably between individuals, suggesting the involvement of mechanistic co-factors and potential for therapeutically beneficial interventions. We show in Hepa1.6 cells that the mildly polymerogenic I (Arg39Cys) AAT mutant forms aberrant inter- and intra-molecular disulphide bonds involving the acquired Cys39 and the only cysteine residue in the wild-type (M) sequence (Cys232). Substitution of Cys39 to serine partially restores secretion, showing that disulphide bonding contributes to the intracellular retention of I AAT. Covalent homodimers mediated by inter-Cys232 bonding alone are also observed in cells expressing the common Z and other polymerising AAT variants where conformational behaviour is abnormal, but not in those expressing M AAT. Prevention of such disulphide linkage through the introduction of the Cys232Ser mutation or by treatment of cells with reducing agents increases Z AAT secretion. Our results reveal that disulphide interactions enhance intracellular accumulation of AAT mutants and implicate the oxidative ER state as a pathogenic co-factor. Redox modulation, e.g. by anti-oxidant strategies, may therefore be beneficial in AAT deficiency-associated liver disease.

  2. Effect of expectoration on inflammation in induced sputum in alpha-1-antitrypsin deficiency.

    PubMed

    Gompertz, Simon; Hill, Adam T; Bayley, Darren L; Stockley, Robert A

    2006-06-01

    It is unclear how chronic expectoration influences airway inflammation in patients with chronic lung disease. The aim of this study was to investigate factors influencing inflammation in induced sputum samples, including, in particular, chronic sputum production. Myeloperoxidase, interleukin-8, leukotriene B4 (LTB4), neutrophil elastase, secretory leukoprotease inhibitor (SLPI) and protein leakage were compared in induced sputum samples from 48 patients (36 with chronic expectoration) with COPD (with and without alpha-1-antitrypsin deficiency; AATD), 9 individuals with AATD but without lung disease and 14 healthy controls. There were no differences in inflammation in induced sputum samples from healthy control subjects and from AATD deficient patients with normal lung function but without chronic expectoration (P>0.05). Inflammation in induced sputum from AATD patients with airflow obstruction and chronic sputum expectoration was significantly greater than for similar patients who did not expectorate: Interleukin-8 (P<0.01), elastase activity (P=0.01), and protein leakage (P<0.01). The presence of spontaneous sputum expectoration in AATD patients with airflow obstruction was associated with increased neutrophilic airway inflammation in induced sputum samples. The presence of chronic expectoration in some patients will clearly complicate interpretation of studies employing sputum induction where this feature has not been identified.

  3. Barriers to genetic testing among persons at risk for alpha-1 antitrypsin deficiency.

    PubMed

    Dickson, Marguerite R; Carter, Cindy L; Carpenter, Matthew J; McClure, Rebecca L; McGee, Dawn A; Zapka, Jane G; Strange, Charlie

    2008-12-01

    The alpha coded testing (ACT) study offers free and confidential testing for alpha-1 antitrypsin deficiency (AATD) and includes surveys to provide data to study the psychosocial correlates of genetic testing. The purpose of the current study is to better understand reasons why some individuals complete genetic testing while others do not. Survey measures were compared between participants who requested and returned a genetic test for AATD (n = 703), and a random sample of individuals who requested a test kit, but did not return it within 3 months of their request (n = 83). Increasing decile of age (odds ratio [OR] = 0.74 [95% confidence interval = 0.60-0.82]) and fingerstick fear (OR = 0.74 [0.60-0.93]) were associated with a decreased likelihood of returning the test, while assurance of confidentiality was associated with an increased likelihood (OR = 1.26 [1.01-1.57]) of returning the genetic test. General anxiety as measured by the Beck Anxiety Inventory, family functioning as measured by the general functioning subscale of the Family Assessment Device, and stress induced by genetic testing as measured by the Impact of Events Scale did not significantly differ between responder groups (p = not significant). Results of this study help characterize factors driving genetic testing in AATD and may offer insight into population responses with other genetic tests.

  4. Intrapulmonary vascular remodeling: MSCT-based evaluation in COPD and alpha-1 antitrypsin deficient subjects

    NASA Astrophysics Data System (ADS)

    Crosnier, Adeline; Fetita, Catalin; Thabut, Gabriel; Brillet, Pierre-Yves

    2016-03-01

    Whether COPD is generally known as a small airway disease, recent investigations suggest that vascular remodeling could play a key role in disease progression. This paper develops a specific investigation framework in order to evaluate the remodeling of the intrapulmonary vascular network and its correlation with other image or clinical parameters (emphysema score or FEV1) in patients with smoking- or genetic- (alpha-1 antitrypsin deficiency - AATD) related COPD. The developed approach evaluates the vessel caliber distribution per lung or lung region (upper, lower, 10%- and 20%- periphery) in relation with the severity of the disease and computes a remodeling marker given by the area under the caliber distribution curve for radii less than 1.6mm, AUC16. It exploits a medial axis analysis in relation with local caliber information computed in the segmented vascular network, with values normalized with respect to the lung volume (for which a robust segmentation is developed). The first results obtained on a 34-patient database (13 COPD, 13 AATD and 8 controls) showed significant vascular remodeling for COPD and AATD versus controls, with a negative correlation with the emphysema degree for COPD, but not for AATD. Significant vascular remodeling at 20% lung periphery was found both for the severe COPD and AATD patients, but not for the moderate groups. Also the vascular remodeling in AATD did not correlate with the FEV1, nor with DLCO, which might suggest independent mechanisms for bronchial and vascular remodeling in the lung.

  5. Elucidation of the molecular logic by which misfolded alpha 1-antitrypsin is preferentially selected for degradation.

    PubMed

    Wu, Ying; Swulius, Matthew T; Moremen, Kelley W; Sifers, Richard N

    2003-07-01

    The exocytic pathway provides a physical route through which newly synthesized secretory and membrane proteins are deployed to the eukaryote cell surface. For newly synthesized alpha1-antitrypsin (AAT), the modification of its asparagine-linked oligosaccharides by a slow-acting mannosidase partitions the misfolded monomer into the proteasomal degradation pathway. Herein, we asked whether, and how, modification by endoplasmic reticulum mannosidase I (ERManI) contributes to the preferential selection of the misfolded AAT monomer for proteasomal degradation. Transiently expressed mutant and WT AAT variants underwent rapid destabilization in response to an artificially elevated ERManI concentration in the murine hepatoma cell line, Hepa1a. Based on the mannosidase- and lactacystin-sensitive properties of intracellular turnover, a stochastic model is proposed in which the delayed onset of the glycan modification, relative to the duration of nonnative protein structure, coordinates the preferential degradation of the misfolded monomer and spares the native molecule from destruction. Newly synthesized endogenous transferrin underwent degradation in response to an elevated concentration of ERManI, whereas the nonglycosylated secretory glycoprotein albumin was not affected. Taken together, these findings indicate that efficient conformational maturation might function as the initial quality control standard for a broad population of glycoproteins.

  6. Alpha1-antitrypsin as model to assess glycan function in endoplasmic reticulum.

    PubMed

    Termine, Daniel; Wu, Ying; Liu, Yan; Sifers, Richard N

    2005-04-01

    It is now understood that a cohesive series of quality control checkpoints ensures the accuracy of gene expression in eukaryotic cells. Although initiated in the nucleus to monitor the integrity of inherited genetic information, the quality control program encompasses post-translational events that facilitate the structural maturation of encoded proteins or target them for degradation if unable to adopt native structure. Given the fact that many genetic mutations actually manifest themselves at the level of aberrant protein structure, a current challenge in the post-genomics era is to elucidate how post-translational checkpoints can modify the severity of numerous loss-of-function and gain-of-toxic-function diseases, possibly influencing an individual's susceptibility toward the development of the associated pathologies. The purpose of this chapter is to describe the experimental methodology by which alpha1-antitrypsin has been used as a molecular reagent to define the mechanisms by which the processing and recognition of asparagine-linked oligosaccharides can orchestrate the fate of newly synthesized glycoproteins in the early secretory pathway. The conceptual framework, and associated techniques, can serve as a roadmap for the investigation of other mutated glycoproteins, many of which can contribute to disease. PMID:15804606

  7. Alpha 1 Antitrypsin Inhibits Dendritic Cell Activation and Attenuates Nephritis in a Mouse Model of Lupus.

    PubMed

    Elshikha, Ahmed S; Lu, Yuanqing; Chen, Mong-Jen; Akbar, Mohammad; Zeumer, Leilani; Ritter, Andrea; Elghamry, Hanaa; Mahdi, Mahmoud A; Morel, Laurence; Song, Sihong

    2016-01-01

    Systemic lupus erythematosus (SLE) is an autoimmune disorder with a worldwide distribution and considerable mortality and morbidity. Although the pathogenesis of this disease remains elusive, over-reactive dendritic cells (DCs) play a critical role in the disease development. It has been shown that human alpha-1 antitrypsin (hAAT) has protective effects in type 1 diabetes and rheumatoid arthritis mouse models. In the present study, we tested the effect of AAT on DC differentiation and functions, as well as its protective effect in a lupus-prone mouse model. We showed that hAAT treatment significantly inhibited LPS (TLR4 agonist) and CpG (TLR9 agonist) -induced bone-marrow (BM)-derived conventional and plasmacytoid DC (cDC and pDC) activation and reduced the production of inflammatory cytokines including IFN-I, TNF-α and IL-1β. In MRL/lpr mice, hAAT treatment significantly reduced BM-derived DC differentiation, serum autoantibody levels, and importantly attenuated renal pathology. Our results for the first time demonstrate that hAAT inhibits DC activation and function, and it also attenuates autoimmunity and renal damage in the MRL/lpr lupus model. These results imply that hAAT has a therapeutic potential for the treatment of SLE in humans. PMID:27232337

  8. Conductivity in Exhaled Breath Condensate from Subjects with Emphysema and Type ZZ alpha-1-Antitrypsin Deficiency.

    PubMed

    Stolk, Jan; Fumagalli, Marco; Viglio, Simona; Iadarola, Paolo

    2015-05-01

    The assessment of biomarkers in biological samples from the lung has long been employed. Upon cooling water vapor present in exhaled breath, variable amounts of droplets of condensate (EBC) containing volatile and non-volatile compounds may be easily and non-invasively obtained from patients of any age.Objective of the present study was to compare the level of EBC conductivity determined for cohorts of individuals with different inflammatory lung disorders with that of healthy never-smoking individuals.The conductivity in EBC of PiZZ-Alpha-1-antitrypsin deficiency patients with a diagnosis of emphysema (PiZZ-AATD) was 3 fold lower than in spouse controls (54.5 ± 11.6 vs 165.3 ± 10.7 μS/cm). Non-PiZZ emphysema patients had conductivity in EBC of 59.6 ± 5.8 μS/cm and patients with sarcoidosis without airflow obstruction had EBC conductivity of 178,8 ± 6,2 μS/cm, 
not significantly different (p = 0.5) from healthy controls. Conductivity in serial EBC samples from patients with PiZZ-AATD emphysema and healthy controls was stable in 6 different samples collected over a period of 14 months. We conclude that conductivity values in EBC can be used as a correction factor for dilution of non-volatile components in EBC.

  9. [Alpha-1 Antitrypsin Affects U0126-Induced Cytotoxicity in Colon Cancer Cell Line (HCT116)].

    PubMed

    Ljujic, M; Mijatovic, S; Bulatovic, M Z; Mojic, M; Maksimovic-Ivanic, D; Radojkovic, D; Topic, A

    2016-01-01

    Alpha-1-antitrypsin (AAT), an acute phase protein, is the principal circulatory anti-protease. This multifunctional protein is encoded by the SERPINA1 gene. Although AAT was recognised as a potential tumour marker, its role in cancer biology remains unknown. Given that it has been demonstrated that AAT has an anti-apoptotic property against non-malignant cells, we aimed to investigate whether AAT affects apoptosis in a colon cancer cell line (HCT116). The presence of AAT in the HCT116 cell culture antagonized cytotoxicity of blockers of MEK1/2, PI3K/Akt pathways as well as NF-κB. The dominantly recovered cell viability was observed in the co-treatment with MEK1/2 inhibitor U0126. In addition, it was revealed that AAT almost completely abolished U0126-induced apoptosis through maintenance of the autophagy process. Our study revealed for the first time that the observed cyto-protection triggered by AAT was accompanied by sustained autophagy which opposed apoptosis. These results may contribute to understanding of the role of AAT in cancer development and evaluation of efficacy of cancer therapy.

  10. Lung clearance index for monitoring early lung disease in alpha-1-antitrypsin deficiency.

    PubMed

    Fuchs, Susanne I; Schwerk, Nicolaus; Pittschieler, Klaus; Ahrens, Frank; Baden, Winfried; Bals, Robert; Fähndrich, Sebastian; Gleiber, Wolfgang; Griese, Matthias; Hülskamp, Georg; Köhnlein, Thomas; Reckling, Ludmilla; Rietschel, Ernst; Staab, Doris; Gappa, Monika

    2016-07-01

    Patients with alpha-1-antitrypsin deficiency (AATD) and a PI-ZZ genotype are at high risk to develop severe emphysema during adulthood. However, little is known about early stages of emphysema and disease manifestation in other PI-types. Spirometry is commonly used for monitoring although early manifestation of emphysema is suspected within the peripheral airways that are not accessible by forced expiratory manoeuvres. We hypothesized that the Lung Clearance Index (LCI) derived from multiple breath nitrogen-washout (N2-washout) is useful to bridge this diagnostic gap. Patients from age 4 years onward and different PI-types performed N2-washout and spirometry. Results were compared to controls. 193 patients (4-79 years, 75% PI-ZZ) and 33 controls (8-60 years) were included. Mean (SD) LCI in patients was 9.1 (3.1) and 6.3 (0.6) in controls (p ≤ 0.001). 47% of adult patients with other than PI-ZZ genotypes and 39% of all patients with normal spirometry had abnormal LCIs. The LCI measured by N2-washout discriminates between patients with AATD and controls, reflects AATD related lung disease in all stages and appears to identify early peripheral lung changes in younger age than spirometry. We conclude that a normal spirometry does not exclude presence of AATD related lung disease even in genotypes other than PI-ZZ. PMID:27296827

  11. Genetic diversity of the alpha-1-antitrypsin gene in Africans identified using a novel genotyping assay.

    PubMed

    Hayes, Vanessa M

    2003-07-01

    The highly polymorphic human alpha-1-antitrypsin (AAT) gene, more recently named SERPINA1, codes for the most abundant circulating plasma serine protease inhibitor, protease inhibitor 1 (PI). Most studies determining AAT haplotype frequencies have been restricted first by the limited accuracy of the phenotypic method used and secondly by the analysis of predominantly Caucasian populations. Limited studies have been performed on African-based populations. Here a new comprehensive assay for genotyping the entire coding region, including splice junctions, of the AAT gene was designed. This assay, based on denaturing gradient gel electrophoresis (DGGE), allows for the complete analysis of a single individual in two lanes on a gel. Application of the assay resulted in the identification of nine known AAT variants as well as 13 novel sequence variants, five of which are single nucleotide polymorphisms (SNPs), occurring exclusively in the African-based populations. This is the first comprehensive analysis of the genetic diversity of the AAT gene in a cohort from sub-Saharan Africa.

  12. The contribution of the conserved hinge region residues of alpha1-antitrypsin to its reaction with elastase.

    PubMed

    Hopkins, P C; Stone, S R

    1995-12-01

    The hinge region of serpins is a conserved sequence of 8 amino acids located 7 residues away from the scissile bond at P8 to P15, on the edge of the protease-binding domain. In the inhibitory serpins the P8 to P12 residues of this motif are usually small side-chain amino acids, most commonly alanine. Each of these residues in alpha1-antitrypsin was mutated to a glutamate, and the effect of a hinge-region glutamic acid substitution was found. While substitutions at positions P10 and P12 affected the inhibitory characteristics of alpha1-antitrypsin, substitutions at positions P7, P8, P9, and P11 had no effect on inhibition. Thus, the conservation of residues with small side chains at the latter positions does not appear to be related to an essential function in the inhibitory mechanism. Following the glutamate substitution at P10, alpha1-antitrypsin remained a rapid inhibitor of elastase, but the elastase--serpin complex slowly broke down to yield active elastase and cleaved alpha1-antitrypsin. The glutamate substitution at P12 caused the resultant molecule (P12 Ala-->Glu) to become a partial substrate of elastase such that four moles of inhibitor were required to inhibit one mole of enzyme, and led to a 12-fold decrease in the association rate constant. The data could be interpreted in terms of the suicide substrate inhibition model for serpin-protease interactions and allowed a further refinement of the role of the hinge region in this process.

  13. [Uniform method for determining the alpha 1-antitrypsin and alpha 2-macroglobulin activity in human blood serum (plasma)].

    PubMed

    Nartikova, V F; Paskhina, T S

    1979-01-01

    A modified spectrophotometric method is developed for simultaneous estimation of alpha 1-antitrypsin and alpha 2-macroglobulin in human blood serum (plasma); the method is based on dissimilar interaction of these inhibitors with trypsin in the systems with a low molecular substrate N-alpha-benzoyl-l-arginine ethyl ester. alpha 1-Antitrypsin was estimated by inhibition of the arginine esterase activity of trypsin in a mixture containing human blood serum diluted 50-fold. alpha 2-Macroglobulin was estimated by maintained arginine esterase activity of the trypsin-alpha 2-macroglobulin complex, formed after interaction of an excess of trypsin with blood serum, diluted 10-fold and after subsequent inactivation of free, unbound with alpha 2-macroglobulin, trypsin by treatment with the soy bean inhibitor of trypsin. alpha 1-Antitrypsin and alpha 2-macrog-obulin were estimated by means of the method described in blood serum of healthy persons and in patients with burns or with carcinoma of pancreas. The method enables to estimate two main inhibitors of blood plasma proteinases in a small volume of blood serum (0.1 ml) very rapidly and specifically using commercially available substrate; the method might be recommended for routine clinical analysis.

  14. Concerted regulation of inhibitory activity of alpha 1-antitrypsin by the native strain distributed throughout the molecule.

    PubMed

    Seo, Eun Joo; Lee, Cheolju; Yu, Myeong-Hee

    2002-04-19

    The native forms of common globular proteins are in their most stable state but the native forms of plasma serpins (serine protease inhibitors) show high energy state interactions. The high energy state strain of alpha(1)-antitrypsin, a prototype serpin, is distributed throughout the whole molecule, but the strain that regulates the function directly appears to be localized in the region where the reactive site loop is inserted during complex formation with a target protease. To examine the functional role of the strain at other regions of alpha(1)-antitrypsin, we increased the stability of the molecule greatly via combining various stabilizing single amino acid substitutions that did not affect the activity individually. The results showed that a substantial increase of stability, over 13 kcal mol(-1), affected the inhibitory activity with a correlation of 11% activity loss per kcal mol(-1). Addition of an activity affecting single residue substitution in the loop insertion region to these very stable substitutions caused a further activity decrease. The results suggest that the native strain of alpha(1)-antitrypsin distributed throughout the molecule regulates the inhibitory function in a concerted manner. PMID:11834734

  15. Reduction of the elastase inhibitory capacity of alpha 1-antitrypsin by peroxides in cigarette smoke: an analysis of brands and filters

    SciTech Connect

    Cohen, A.B.; James, H.L.

    1982-07-01

    A procedure for measuring the oxidant content of aqueous condensates of tobacco cigarette smoke is described. The procedure was used in conjunction with analysis of the ability of the smoke solutions to inactivate the elastase inhibitory capacity (EIC) of alpha 1-antitrypsin. The ability of the smoke of a brand to inactivate alpha 1-antitrypsin correlates well with the known tar and nicotine and with the amount of oxidants as measured using o-dianisidine. Filters were found to remove about 73% of the oxidants from smoke. Smoke from a commercial nontobacco cigarette was also found to contain a significant amount of oxidants and to also destroy alpha 1-antitrypsin. Catalase and superoxide dismutase reduce the effect of solutions containing smoke on the EIC of alpha 1-antitrypsin, suggesting that peroxides and superoxide anions in smoke contribute to the oxidant capacity of the smoke. The extent of apparent oxidation by a given quantity of smoke condensate increases for as long as an hour from the time the condensate is collected. The addition of hydrogen peroxide to the smoke solution increases both its oxidant content and its ability to inactivate alpha 1-antitrypsin. These data suggest that occurrence of hydrogen peroxide caused by secretion from macrophages found in the small airways of smokers may contribute to a locally damaging environment for alpha 1-antitrypsin in the presence of cigarette smoke that could promote the development of centrilobular emphysema.

  16. Reduction of the elastase inhibitory capacity of alpha 1-antitrypsin by peroxides in cigarette smoke: an analysis of brands and filters.

    PubMed

    Cohen, A B; James, H L

    1982-07-01

    A procedure for measuring the oxidant content of aqueous condensates of tobacco cigarette smoke is described. The procedure was used in conjunction with analysis of the ability of the smoke solutions to inactivate the elastase inhibitory capacity (EIC) of alpha 1-antitrypsin. The ability of the smoke of a brand to inactivate alpha 1-antitrypsin correlates well with the known tar and nicotine and with the amount of oxidants as measured using o-dianisidine. Filters were found to remove about 73% of the oxidants from smoke. Smoke from a commercial nontobacco cigarette was also found to contain a significant amount of oxidants and to also destroy alpha 1-antitrypsin. Catalase and superoxide dismutase reduce the effect of solutions containing smoke on the EIC of alpha 1-antitrypsin, suggesting that peroxides and superoxide anions in smoke contribute to the oxidant capacity of the smoke. The extent of apparent oxidation by a given quantity of smoke condensate increases for as long as an hour from the time the condensate is collected. The addition of hydrogen peroxide to the smoke solution increases both its oxidant content and its ability to inactivate alpha 1-antitrypsin. These data suggest that occurrence of hydrogen peroxide caused by secretion from macrophages found in the small airways of smokers may contribute to a locally damaging environment for alpha 1-antitrypsin in the presence of cigarette smoke that could promote the development of centrilobular emphysema. PMID:6979963

  17. Efficacy of alpha1-antitrypsin augmentation therapy in conditions other than pulmonary emphysema.

    PubMed

    Blanco, Ignacio; Lara, Beatriz; de Serres, Frederick

    2011-04-12

    Up to now alpha 1-antitrypsin (AAT) augmentation therapy has been approved only for commercial use in selected adults with severe AAT deficiency-related pulmonary emphysema (i.e. PI*ZZ genotypes as well as combinations of Z, rare and null alleles expressing AAT serum concentrations <11 μmol/L). However, the compassionate use of augmentation therapy in recent years has proven outstanding efficacy in small cohorts of patients suffering from uncommon AAT deficiency-related diseases other than pulmonary emphysema, such as fibromyalgia, systemic vasculitis, relapsing panniculitis and bronchial asthma. Moreover, a series of preclinical studies provide evidence of the efficacy of AAT augmentation therapy in several infectious diseases, diabetes mellitus and organ transplant rejection. These facts have generated an expanding number of medical applications and patents with claims for other indications of AAT besides pulmonary emphysema. The aim of the present study is to compile and analyze both clinical and histological features of the aforementioned published case studies and reports where AAT augmentation therapy was used for conditions other than pulmonary emphysema. Particularly, our research refers to ten case reports and two clinical trials on AAT augmentation therapy in patients with both AAT deficiency and, at least, one of the following diseases: fibromyalgia, vasculitis, panniculitis and bronchial asthma. In all the cases, AAT was successfully applied whereas previous maximal conventional therapies had failed. In conclusion, laboratory studies in animals and humans as well as larger clinical trials should be, thus, performed in order to determine both the strong clinical efficacy and security of AAT in the treatment of conditions other than pulmonary emphysema.

  18. Chitosan-genipin nanohydrogel as a vehicle for sustained delivery of alpha-1 antitrypsin

    PubMed Central

    Ghasemi, Ahmad; Mohtashami, Mahnaz; Sheijani, Samaneh Sotoudeh; Aliakbari, Kamelya

    2015-01-01

    Alpha-1antitrypsin (A1AT) deficiency, an inherited disorder, has been shown to be the cause of lung diseases such as emphysema and chronic obstructive pulmonary disease. One of the treatment strategies to provide appropriate and adequate concentrations of A1AT in the lungsis the application of nanoparticles (NPs) in pulmonary drug delivery. In the current study, biocompatible nanohydrogels were prepared using chemically cross-linked chitosan with ginepin, a natural cross linker reagent, and used as a carrier to deposit A1AT into the lung tissue. Colloidal and monodispersed NPs were synthesized through reverse microemulsion. Nanohydrogels were characterized with TEM, LLS, FTIR, ZTEA potential, UV spectrum, and swelling test. Encapsulation efficacy was determined at different concentrations of A1AT using Bradford assay. Effect of processing variables such as pH, loading efficiency, and release media components on drug release profile was determined in simulated lung fluids. To evaluate the inhibitory activity of the A1AT after release from NPs, trypsin inhibitory capacity assay was carried out. Results from FTIR and UV spectrum confirmed the development of chitosan cross linkage. Spherical chitosan-genipin NPs were sized from 30-100 nm. NPs exhibited the ability to release 49% of the drug within 12-dayperiodatpH 7. However, there were variations with the drug release profile due to pH variations and loading efficacy. Drug release was higher in pseudo alveolar fluid in comparison with saline solution. These data indicate that application of chitosan nanohydrogels can be a useful tool for sustained release of A1AT in the lung tissue. PMID:26779272

  19. Alpha1-Antitrypsin Attenuates Renal Fibrosis by Inhibiting TGF-β1-Induced Epithelial Mesenchymal Transition.

    PubMed

    Cho, Jang-Hee; Ryu, Hye-Myung; Oh, Eun-Joo; Yook, Ju-Min; Ahn, Ji-Sun; Jung, Hee-Yeon; Choi, Ji-Young; Park, Sun-Hee; Kim, Yong-Lim; Kwak, Ihm Soo; Kim, Chan-Duck

    2016-01-01

    Alpha1-antitrypsin (AAT) exerts its anti-inflammatory effect through regulating the activity of serine proteinases. This study evaluated the inhibitory effects of AAT against the transforming growth factor (TGF)-β1 induced epithelial-to-mesenchymal transition (EMT) in unilateral ureter obstruction (UUO) mice and Madin-Darby canine kidney (MDCK) cells. C57BL/6 mice with induced UUO were injected intraperitoneally with AAT (80 mg/Kg) or vehicle for 7 days. MDCK cells were treated with TGF-β1 (2 ng/mL) for 48 hours to induce EMT, and co-treated with AAT (10 mg/mL) to inhibit the EMT. Masson's trichrome and Sirius red staining was used to estimate the extent of renal fibrosis in UUO mice. The expression of alpha-smooth muscle actin (α-SMA), vimentin, fibronectin, collagen I, and E-cadherin in MDCK cells and kidney tissue were evaluated. Masson's and Sirius red staining revealed that the area of renal fibrosis was significantly smaller in AAT treated UUO group compared with that of UUO and vehicle treated UUO groups. AAT treatment attenuated upregulation of Smad2/3 phosphorylation in UUO mouse model. Co-treatment of MDCK cells with TGF-β1 and AAT significantly attenuated the changes in the expression of α-SMA, vimentin, fibronectin, collagen I, and E-cadherin. AAT also decreased the phosphorylated Smad3 expression and the phosphorylated Smad3/Smad3 ratio in MDCK cells. AAT treatment inhibited EMT induced by TGF-β1 in MDCK cells and attenuated renal fibrosis in the UUO mouse model. The results of this work suggest that AAT could inhibit the process of EMT through the suppression of TGF-β/Smad3 signaling. PMID:27607429

  20. Alpha-1 antitrypsin is markedly decreased following pulmonary F. tularensis challenge.

    PubMed

    Chambers, James P; Yu, Jieh-Juen; Jupelli, Madhulika; Weintraub, Susan T; Lopez-Ribot, Jose L; Valdes, James J; Arulanandam, Bernard P

    2011-01-01

    Neutrophils form the first line of defense during infection and are indispensable in this function. The neutrophil elastase is a key effector molecule of the innate immune system with potent antimicrobial activity against Gram-negative bacteria, spirochaetes, and fungi. However, the release of neutrophil elastase during bacterial infection must be checked otherwise its release in the extracellular milieu will result in damage to surrounding tissues. Alpha-1 antitrypsin is a small glycoprotein clade A serpine serine protease inhibitor and has been shown to increase in humans following bacterial and viral infection. Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of tularemia. Type A strains are the most virulent with an infectious dose as low as 10 colony forming units and a mortality rate of 30-60% among untreated cases of pneumonic tularemia. We report here significant reduction of this major inhibitor of the neutrophil elastase in plasma of F. tularensis LVS and F. tularensis (type A) SCHU S4 infected animals following pulmonary challenge. Associated with an imbalance of protease-antiprotease function at the alveolar level in lungs of infected animals, increased elastase activity was observed in lung lavage fluids accompanied by decrease lung function, i.e., loss of lung elastance with concomitant increase of pulmonary hysteresivity. Consistent with a competent acute phase response following F. tularensis LVS and F. tularensis (type A) SCHU S4 pulmonary challenge and proposed up-regulation of plasma haptoglobin during the course of the acute phase reaction, haptoglobin was observed significantly increased. These data suggest that unchecked neutrophil serine protease activity may arise from F. tularensis targeted reduction of plasma α(1)-antitrysin promoting lung tissue damage facilitating increased dissemination of this bacterium in infected animals. PMID:22919586

  1. Alpha1-Antitrypsin Attenuates Renal Fibrosis by Inhibiting TGF-β1-Induced Epithelial Mesenchymal Transition

    PubMed Central

    Cho, Jang-Hee; Ryu, Hye-Myung; Oh, Eun-Joo; Yook, Ju-Min; Ahn, Ji-Sun; Jung, Hee-Yeon; Choi, Ji-Young; Park, Sun-Hee; Kim, Yong-Lim; Kwak, Ihm Soo; Kim, Chan-Duck

    2016-01-01

    Alpha1-antitrypsin (AAT) exerts its anti-inflammatory effect through regulating the activity of serine proteinases. This study evaluated the inhibitory effects of AAT against the transforming growth factor (TGF)-β1 induced epithelial-to-mesenchymal transition (EMT) in unilateral ureter obstruction (UUO) mice and Madin-Darby canine kidney (MDCK) cells. C57BL/6 mice with induced UUO were injected intraperitoneally with AAT (80 mg/Kg) or vehicle for 7 days. MDCK cells were treated with TGF-β1 (2 ng/mL) for 48 hours to induce EMT, and co-treated with AAT (10 mg/mL) to inhibit the EMT. Masson’s trichrome and Sirius red staining was used to estimate the extent of renal fibrosis in UUO mice. The expression of alpha-smooth muscle actin (α-SMA), vimentin, fibronectin, collagen I, and E-cadherin in MDCK cells and kidney tissue were evaluated. Masson’s and Sirius red staining revealed that the area of renal fibrosis was significantly smaller in AAT treated UUO group compared with that of UUO and vehicle treated UUO groups. AAT treatment attenuated upregulation of Smad2/3 phosphorylation in UUO mouse model. Co-treatment of MDCK cells with TGF-β1 and AAT significantly attenuated the changes in the expression of α-SMA, vimentin, fibronectin, collagen I, and E-cadherin. AAT also decreased the phosphorylated Smad3 expression and the phosphorylated Smad3/Smad3 ratio in MDCK cells. AAT treatment inhibited EMT induced by TGF-β1 in MDCK cells and attenuated renal fibrosis in the UUO mouse model. The results of this work suggest that AAT could inhibit the process of EMT through the suppression of TGF-β/Smad3 signaling. PMID:27607429

  2. Alpha-1 Antitrypsin is Markedly Decreased Following Pulmonary F. tularensis Challenge

    PubMed Central

    Chambers, James P.; Yu, Jieh-Juen; Jupelli, Madhulika; Weintraub, Susan T.; Lopez-Ribot, Jose L.; Valdes, James J.; Arulanandam, Bernard P.

    2011-01-01

    Neutrophils form the first line of defense during infection and are indispensable in this function. The neutrophil elastase is a key effector molecule of the innate immune system with potent antimicrobial activity against Gram-negative bacteria, spirochaetes, and fungi. However, the release of neutrophil elastase during bacterial infection must be checked otherwise its release in the extracellular milieu will result in damage to surrounding tissues. Alpha-1 antitrypsin is a small glycoprotein clade A serpine serine protease inhibitor and has been shown to increase in humans following bacterial and viral infection. Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of tularemia. Type A strains are the most virulent with an infectious dose as low as 10 colony forming units and a mortality rate of 30–60% among untreated cases of pneumonic tularemia. We report here significant reduction of this major inhibitor of the neutrophil elastase in plasma of F. tularensis LVS and F. tularensis (type A) SCHU S4 infected animals following pulmonary challenge. Associated with an imbalance of protease–antiprotease function at the alveolar level in lungs of infected animals, increased elastase activity was observed in lung lavage fluids accompanied by decrease lung function, i.e., loss of lung elastance with concomitant increase of pulmonary hysteresivity. Consistent with a competent acute phase response following F. tularensis LVS and F. tularensis (type A) SCHU S4 pulmonary challenge and proposed up-regulation of plasma haptoglobin during the course of the acute phase reaction, haptoglobin was observed significantly increased. These data suggest that unchecked neutrophil serine protease activity may arise from F. tularensis targeted reduction of plasma α1-antitrysin promoting lung tissue damage facilitating increased dissemination of this bacterium in infected animals. PMID:22919586

  3. Faecal alpha-1-antitrypsin and excretion of 111indium granulocytes in assessment of disease activity in chronic inflammatory bowel diseases.

    PubMed Central

    Fischbach, W; Becker, W; Mössner, J; Koch, W; Reiners, C

    1987-01-01

    Intestinal protein loss in chronic inflammatory bowel diseases may be easily determined by measurement of alpha-1-antitrypsin (alpha 1-AT) stool concentration and alpha 1-AT clearance. Both parameters were significantly raised in 36 and 34 patients respectively with chronic inflammatory bowel diseases, compared with eight patients with non-inflammatory bowel diseases, or 19 healthy volunteers. There was wide range of overlap between active and inactive inflammatory disease. Contrary to serum alpha 1-AT, faecal excretion and clearance of alpha 1-AT did not correlate with ESR, serum-albumin, orosomucoid, and two indices of disease activity. A comparison of alpha 1-AT faecal excretion and clearance with the faecal excretion of 111In labelled granulocytes in 27 patients with chronic inflammatory bowel diseases, showed no correlation between the intestinal protein loss and this highly specific marker of intestinal inflammation. Enteric protein loss expressed by faecal excretion and clearance of alpha 1-AT does not depend on mucosal inflammation only, but may be influenced by other factors. PMID:3495470

  4. Why has it been so difficult to prove the efficacy of alpha-1-antitrypsin replacement therapy? Insights from the study of disease pathogenesis

    PubMed Central

    Dickens, Jennifer A; Lomas, David A

    2011-01-01

    Alpha-1-antitrypsin is the most abundant circulating protease inhibitor. It is mainly produced by the liver and secreted into the circulation where it acts to prevent excessive proteolytic damage in the lungs by the enzyme neutrophil elastase. The most common severe deficiency allele is the Z mutation, which causes the protein to self-associate into ordered polymers. These polymers accumulate within hepatocytes to cause liver damage. The resulting lack of circulating α1-antitrypsin predisposes the Z homozygote to proteolytic lung damage and emphysema. Other pathways may also contribute to the development of lung disease. In particular, polymers of Z α1-antitrypsin can form within the lung where they act as a pro-inflammatory stimulus that may exacerbate protease-mediated lung damage. Researchers recognized in the 1980s that plasma α1-antitrypsin levels could be restored by intravenous infusions of purified human protein. Alpha-1-antitrypsin replacement therapy was introduced in 1987 but subsequent clinical trials have produced conflicting results, and to date there remains no widely accepted clinical evidence of the efficacy of α1-antitrypsin replacement therapy. This review addresses our current understanding of disease pathogenesis in α1-antitrypsin deficiency and questions why this treatment in isolation may not be effective. In particular it discusses the possible role of α1-antitrypsin polymers in exacerbating intrapulmonary inflammation and attenuating the efficacy of α1-antitrypsin replacement therapy. PMID:21966212

  5. Individualized lung function trends in alpha-1-antitrypsin deficiency: a need for patience in order to provide patient centered management?

    PubMed Central

    Stockley, Robert A; Edgar, Ross G; Pillai, Anilkumar; Turner, Alice M

    2016-01-01

    Background Chronic obstructive pulmonary disease (COPD) is characterized by fixed airflow obstruction and accelerated decline of forced expired volume in 1 second (FEV1). Alpha-1-antitrypsin deficiency is a genetic cause of COPD and associated with more rapid decline in lung function, even in some never smokers (NS) but the potential for individualized assessment to reveal differences when compared to group analyses has rarely been considered. Methods We analyzed decline in post-bronchodilator FEV1 and gas transfer (% predicted) over at least 3 years (mean= 6.11, 95% CI 5.80–6.41) in our unique data set of 482 patients with alpha-1-antitrypsin deficiency (PiZ) to determine individual rates of decline, implications for prognosis, and potential clinical management. Findings There was a marked variation in individual rates of FEV1 decline from levels consistent with normal aging (observed in 23.5% of patients with established COPD, 57.5% of those without) to those of rapidly declining COPD. Gas transfer did not decline in 12.8% of NS and 20.7% of ex-smokers with established COPD (33.3% and 25.0%, respectively, for those without COPD). There was no correlation between decline in gas transfer and FEV1 for those with COPD, although a weak relationship existed for those without (r=0.218; P<0.025). Conclusion These data confirm differing individual rates of lung function decline in alpha-1-antitrypsin deficiency, indicating the importance of comprehensive physiological assessment and a personalized approach to patient management. PMID:27536086

  6. Linkage between the variegate porphyria (VP) and the alpha-1-antitrypsin (PI) genes on human chromosome 14.

    PubMed

    Bissbort, S; Hitzeroth, H W; du Wentzel, D P; Van den Berg, C W; Senff, H; Wienker, T F; Bender, K

    1988-07-01

    From family studies close linkage between the gene locus for variegate porphyria (VP) and the alpha-1-antitrypsin (PI) gene became evident. The maximal lod score from male meioses was 4.33 at theta = 0.04 and from both sexes combined 3.56 at theta = 0.12. Three pedigrees were triple informative regarding loci VP, PI, and IGHC (immunoglobulin heavy chain cluster, Gm polymorphism). In two of the respective meioses recombinations were observed, and in both cases the co-segregating VP and PI alleles were separated from the Gm haplotypes. These findings argue in favour of gene order either VP:PI:IGHC or PI:VP:IGHC.

  7. Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and. alpha. 1-antitrypsin genes

    SciTech Connect

    Costa, R.H. ); Grayson, D.R. ); Darnell, J.E. Jr. )

    1989-04-01

    Transthyretin (TTR) and {alpha}1-antitrypsin ({alpha}1-AT) are expressed at high levels in the liver and also in at least one other cell type. The authors report here a detailed analysis of the proximal regulatory region of the TTR gene, which has uncovered two new DNA-binding factors that are present mainly (or only) in hepatocytes. One of these new factors, hepatocyte nuclear factor 3 (HNF-3), binds to two sites that are crucial in TTR expression as well as to two additional sites in the {alpha}1-AT proximal enhancer region. The second new factor, HNF-4, binds to two sites in TTR that are required for gene activity. The authors had previously identified binding sites for another hepatocyte-enriched DNA-binding protein (C/EBP or a relative thereof), and additional promoter-proximal sites for that protein in both TTR and {alpha}1-AT are also reported here. From these results it seems clear that cell-specific expression is not simply the result of a single cell-specific factor for each gene but the results of a combination of such factors. The variation and distribution of such factors among different cell types could be an important basis for the coordinate expression of the TTR and {alpha}1-AT genes in the liver or the discordant transcriptional activation of these genes in a few other cell types. The identification of such cell-enriched factors is a necessary prelude to understanding the basis for cell specificity.

  8. Alpha 1-antitrypsin Pittsburgh (Met358-->Arg) inhibits the contact pathway of intrinsic coagulation and alters the release of human neutrophil elastase during simulated extracorporeal circulation.

    PubMed

    Wachtfogel, Y T; Bischoff, R; Bauer, R; Hack, C E; Nuijens, J H; Kucich, U; Niewiarowski, S; Edmunds, L H; Colman, R W

    1994-12-01

    Cardiopulmonary bypass prolongs bleeding time, increases postoperative blood loss, and triggers activation of plasma proteolytic enzyme systems and blood cells referred to as the "whole body inflammatory response". Contact of blood with synthetic surfaces leads to qualitative and quantitative alterations in platelets, neutrophils, contact and complement systems. Contact and complement pathway proteins both induce neutrophil activation. alpha 1-antitrypsin Pittsburgh (Met358-->Arg), a mutant of alpha 1-antitrypsin, is a potent inhibitor of plasma kallikrein and thrombin. We investigated whether this recombinant mutant protein inhibited platelet activation, as well as contact and/or complement-induced neutrophil activation during simulated extracorporeal circulation. Arg358 alpha 1-antitrypsin did not prevent the 34% drop in platelet count at 5 min of recirculation, did not block the 50% decrease in ADP-induced platelet aggregation at 120 min of recirculation, nor inhibit the release of 6.06 +/- 1.07 micrograms/ml beta-thromboglobulin at 120 min of recirculation suggesting that the inhibitor had little effect on platelet activation. However, Arg358 alpha 1-antitrypsin totally blocked kallikrein-C1-inhibitor complex formation but not C1-C1-inhibitor complex formation. Most importantly, Arg358 alpha 1-antitrypsin decreased the release of 1.11 +/- 0.16 micrograms/ml human neutrophil elastase by 43%. The attenuation of neutrophil activation in the absence of an effect on complement activation via the classical pathway, supports the concept that kallikrein is a major mediator of neutrophil degranulation during cardiopulmonary bypass.

  9. Determination of proteinase 3-alpha 1-antitrypsin complexes in inflammatory fluids.

    PubMed

    Dolman, K M; van de Wiel, B A; Kam, C M; Abbink, J J; Hack, C E; Sonnenberg, A; Powers, J C; von dem Borne, A E; Goldschmeding, R

    1992-12-14

    Physiological inhibitors were tested for their in vitro interaction with neutrophil proteinase 3 (PR3). The major plasma proteinase inhibitor of PR3 is alpha 1AT. We have developed a radioimmunoassay (RIA) for quantitative detection of PR3-alpha 1AT complexes formed in vivo in inflammatory exudates such as synovial fluid and plasma from patients with sepsis. Levels of PR3-alpha 1AT complexes correlated significantly with levels of human neutrophil elastase (HNE)-alpha 1AT complexes. Thus, in vivo alpha 1AT not only protects against excessive HNE activity, but also against excessive PR3 activity.

  10. Prevalence of PI*Z and PI*S alleles of alpha-1-antitrypsin deficiency in Finland.

    PubMed

    Häggblom, Jan; Kettunen, Kaisa; Karjalainen, Jussi; Heliövaara, Markku; Jousilahti, Pekka; Saarelainen, Seppo

    2015-01-01

    The prevalence of PI*Z and PI*S alleles of SERPINA1 gene related to alpha-1-antitrypsin deficiency has previously been estimated to be lower in Finland than in the other countries of Northern Europe. The prevalence of PI*M (Malton) has not been studied in Finland before. We determined alpha-1-antitrypsin PI*Z and PI*S and PI*M (Malton) genotypes from a representative population sample. The number of subjects was 6,354 in the PI*S and PI*M (Malton) genotyping. PI*Z genotyping was performed in a subsample of 2,482 subjects. The allele frequencies were PI*Z 19.7/1,000 and PI*S 10.2/1,000. No PI*M (Malton) was found. The number of carriers of PI*Z and PI*S is significantly higher than previously estimated. The prevalences are in line with the findings in the neighboring countries.

  11. Art, alpha-1-antitrypsin polymorphisms and intense creative energy: blessing or curse?

    PubMed

    Schmechel, Donald Everett

    2007-09-01

    Persons heterozygous for Z, S and rare alpha-1-antitrypsin (AAT, SERPIN1A) polymorphisms (ca. 9% of population) are often considered 'silent' carriers with increased vulnerability to environmentally modulated liver and lung disease. They may have significantly more anxiety and bipolar spectrum disorders, nutritional compromise, and white matter disease [Schmechel DE, Browndyke J, Ghio A. Strategies for the dissection of genetic-environmental interactions in neurodegenerative disorders. Neurotoxicology 2006;27:637-57]. Given association of art and mood disorders, we examined occupation and artistic vocation from this same series. One thousand five hundred and thirty-seven consecutive persons aged 16-90 years old received comprehensive work-up including testing for AAT 'phenotype' and level, nutritional factors, and inflammatory, iron and copper indices. Occupations were grouped by Bureau of Labor Standards classification and information gathered on artistic activities. Proportion of reactive airway disease, obstructive pulmonary disease, and pre-existing anxiety disorder or bipolar disorder were significantly increased in persons carrying AAT non-M polymorphisms compared to normal MM genotype (respectively, 10, 20, 21, and 33% compared to 8, 12, 11, and 9%; contingency table, pulmonary: chi2 37, p=0.0001; affective disorder: chi2=171, p=0.0001). In persons with artistic avocation (n=189) or occupation (n=57), AAT non-M polymorphisms are significantly increased (respectively, proportions of 44 and 40% compared to background rate of 9%; contingency table, avocation: chi2=172, p=0.0001; occupation: chi2=57, p=0.0007). Artistic ability and 'anxiety/bipolar spectrum' mood disorders may represent phenotypic attributes that had selective advantage during recent human evolution, an 'intensive creative energy' (ICE) behavioral phenotype. Background proportion of ICE of 7% consists of 49 of 1312 persons with AAT MM genotype (4%), and 58 of 225 persons with non-MM genotypes

  12. Art, alpha-1-antitrypsin polymorphisms and intense creative energy: blessing or curse?

    PubMed

    Schmechel, Donald Everett

    2007-09-01

    Persons heterozygous for Z, S and rare alpha-1-antitrypsin (AAT, SERPIN1A) polymorphisms (ca. 9% of population) are often considered 'silent' carriers with increased vulnerability to environmentally modulated liver and lung disease. They may have significantly more anxiety and bipolar spectrum disorders, nutritional compromise, and white matter disease [Schmechel DE, Browndyke J, Ghio A. Strategies for the dissection of genetic-environmental interactions in neurodegenerative disorders. Neurotoxicology 2006;27:637-57]. Given association of art and mood disorders, we examined occupation and artistic vocation from this same series. One thousand five hundred and thirty-seven consecutive persons aged 16-90 years old received comprehensive work-up including testing for AAT 'phenotype' and level, nutritional factors, and inflammatory, iron and copper indices. Occupations were grouped by Bureau of Labor Standards classification and information gathered on artistic activities. Proportion of reactive airway disease, obstructive pulmonary disease, and pre-existing anxiety disorder or bipolar disorder were significantly increased in persons carrying AAT non-M polymorphisms compared to normal MM genotype (respectively, 10, 20, 21, and 33% compared to 8, 12, 11, and 9%; contingency table, pulmonary: chi2 37, p=0.0001; affective disorder: chi2=171, p=0.0001). In persons with artistic avocation (n=189) or occupation (n=57), AAT non-M polymorphisms are significantly increased (respectively, proportions of 44 and 40% compared to background rate of 9%; contingency table, avocation: chi2=172, p=0.0001; occupation: chi2=57, p=0.0007). Artistic ability and 'anxiety/bipolar spectrum' mood disorders may represent phenotypic attributes that had selective advantage during recent human evolution, an 'intensive creative energy' (ICE) behavioral phenotype. Background proportion of ICE of 7% consists of 49 of 1312 persons with AAT MM genotype (4%), and 58 of 225 persons with non-MM genotypes

  13. Inactivation of alpha 1-antitrypsin by aqueous coal solutions: Possible relation to the emphysema of coal workers

    SciTech Connect

    Huang, X.; Laurent, P.A.; Zalma, R.; Pezerat, H. )

    1993-07-01

    Increasing evidence demonstrates that emphysema in coal workers may be related to their exposure to coal dusts. The hypothesis that emphysema could be related to the production of reactive oxygen species (ROS) generated by inhaled coal dusts was examined in the present study. Using ESR, we investigated whether the interaction of different coals with dissolved oxygen in aqueous medium could generate ROS. Indeed, we found that one of the five examined French coal samples, Vouters coal, was effective in oxidizing formate anions or ethanol by a radical pathway. Inactivation of alpha 1-antitrypsin (alpha 1-AT) in vitro was then examined for all five coal filtrates. The Vouters coal filtrate, which exhibits oxidative activity, can also inactivate alpha 1-AT. When this coal filtrate was crystallized and redissolved, its oxidative activity was found to be conserved. By use of various analytical techniques, the active component of this coal filtrate was identified to be primarily ferrous sulfate. We confirmed that pure ferrous sulfate can effectively reduce oxygen to produce ROS in aqueous medium in vitro and can also inactivate alpha 1-AT. In this report, the nature of the coal-generated oxidative species, the origin of ferrous sulfate, and the stability of ferrous sulfate in the different coal samples are discussed. These results offer evidence that some inhaled coal dusts are capable of producing ROS, which may play an important role in the development of coal workers' emphysema.

  14. Severe postoperative wound healing disturbance in a patient with alpha-1-antitrypsin deficiency: the impact of augmentation therapy.

    PubMed

    Cathomas, Marionna; Schüller, Alexandra; Candinas, Daniel; Inglin, Roman

    2015-10-01

    Wound healing disturbance is a common complication following surgery, but the underlying cause sometimes remains elusive. A 50-year-old Caucasian male developed an initially misunderstood severe wound healing disturbance following colon and abdominal wall surgery. An untreated alpha-1-antitrypsin (AAT) deficiency in the patient's medical history, known since 20 years and clinically apparent as a mild to moderate chronic obstructive pulmonary disease, was eventually found to be at its origin. Further clinical work-up showed AAT serum levels below 30% of the lower reference value; phenotype testing showed a ZZ phenotype and a biopsy taken from the wound area showed the characteristic, disease-related histological pattern of necrotising panniculitits. Augmentation therapy with plasma AAT was initiated and within a few weeks, rapid and adequate would healing was observed. AAT deficiency is an uncommon but clinically significant, possible cause of wound healing disturbances. An augmentation therapy ought to be considered in affected patients during the perioperative period.

  15. Proteome Profiling of Urinary Exosomes Identifies Alpha 1-Antitrypsin and H2B1K as Diagnostic and Prognostic Biomarkers for Urothelial Carcinoma

    PubMed Central

    Lin, Shih-Yi; Chang, Chao-Hsiang; Wu, His-Chin; Lin, Ching-Chan; Chang, Kai-Po; Yang, Chi-Rei; Huang, Chi-Ping; Hsu, Wu-Huei; Chang, Chiz-Tzung; Chen, Chao-Jung

    2016-01-01

    MALDI-TOF spectrometry has not been used for urinary exosome analysis. We used it for determining UC biomarkers. From 2012 to 2015, we enrolled 129 consecutive patients with UC and 62 participants without UC. Exosomes from their urine were isolated, and analyzed through MALDI-TOF spectrometry. Immunohistochemical (IHC) analysis of another 122 UC and 26 non-UC tissues was conducted to verify the discovered biomarkers. Two peaks at m/z 5593 (fragmented peptide of alpha-1-antitrypsin; sensitivity, 50.4%; specificity, 96.9%) and m/z 5947 (fragmented peptide of histone H2B1K sensitivity, 62.0%; specificity, 92.3%) were identified as UC diagnosis exosome biomarkers. UC patients with detectable histone H2B1K showed 2.29- and 3.11-fold increased risks of recurrence and progression, respectively, compared with those with nondetectable histone H2B1K. Verification results of IHC staining revealed significantly higher expression of alpha 1-antitrypsin (p = 0.038) and H2B1K (p = 0.005) in UC tissues than in normal tissues. The expression of alpha 1-antitrypsin and H2B1K in UC tissues was significantly correlated with UC grades (p < 0.05). Urinary exosome proteins alpha 1-antitrypsin and histone H2B1K, which are identified through MALDI-TOF analysis, could facilitate rapid diagnosis and prognosis of UC. PMID:27686150

  16. Gene transfer of master autophagy regulator TFEB results in clearance of toxic protein and correction of hepatic disease in alpha-1-anti-trypsin deficiency

    PubMed Central

    Pastore, Nunzia; Blomenkamp, Keith; Annunziata, Fabio; Piccolo, Pasquale; Mithbaokar, Pratibha; Maria Sepe, Rosa; Vetrini, Francesco; Palmer, Donna; Ng, Philip; Polishchuk, Elena; Iacobacci, Simona; Polishchuk, Roman; Teckman, Jeffrey; Ballabio, Andrea; Brunetti-Pierri, Nicola

    2013-01-01

    Alpha-1-anti-trypsin deficiency is the most common genetic cause of liver disease in children and liver transplantation is currently the only available treatment. Enhancement of liver autophagy increases degradation of mutant, hepatotoxic alpha-1-anti-trypsin (ATZ). We investigated the therapeutic potential of liver-directed gene transfer of transcription factor EB (TFEB), a master gene that regulates lysosomal function and autophagy, in PiZ transgenic mice, recapitulating the human hepatic disease. Hepatocyte TFEB gene transfer resulted in dramatic reduction of hepatic ATZ, liver apoptosis and fibrosis, which are key features of alpha-1-anti-trypsin deficiency. Correction of the liver phenotype resulted from increased ATZ polymer degradation mediated by enhancement of autophagy flux and reduced ATZ monomer by decreased hepatic NFκB activation and IL-6 that drives ATZ gene expression. In conclusion, TFEB gene transfer is a novel strategy for treatment of liver disease of alpha-1-anti-trypsin deficiency. This study may pave the way towards applications of TFEB gene transfer for treatment of a wide spectrum of human disorders due to intracellular accumulation of toxic proteins. PMID:23381957

  17. Alpha1-antitrypsin polymorphism and systematics of eastern North American wolves

    USGS Publications Warehouse

    Mech, L. David; Federoff, Nicholas E.

    2002-01-01

    We used data on the polymorphic status of α1-antitrypsin (α1AT) to study the relationship of Minnesota wolves to the gray wolf (Canis lupus), which was thought to have evolved in Eurasia, and to red wolves (Canis rufus) and coyotes (Canis latrans), which putatively evolved in North America. Recent evidence had indicated that Minnesota wolves might be more closely related to red wolves and coyotes. Samples from wild-caught Minnesota wolves and from captive wolves, at least some of which originated in Alaska and western Canada, were similarly polymorphic for α1AT, whereas coyote and red wolf samples were all monomorphic. Our findings, in conjunction with earlier results, are consistent with the Minnesota wolf being a gray wolf of Eurasian origin or possibly a hybrid between the gray wolf of Eurasian origin and the proposed North American wolf.

  18. Alpha1-antitrypsin polymorphism and systematics of eastern North American wolves

    USGS Publications Warehouse

    Mech, L.D.; Federoff, N.E.; Kueppers, F.

    2002-01-01

    We used data on the polymorphic status of 1-antitrypsin (1AT) to study the relationship of Minnesota wolves to the gray wolf (Canis lupus), which was thought to have evolved in Eurasia, and to red wolves (Canis rufus) and coyotes (Canis latrans), which putatively evolved in North America. Recent evidence had indicated that Minnesota wolves might be more closely related to red wolves and coyotes. Samples from wild-caught Minnesota wolves and from captive wolves, at least some of which originated in Alaska and western Canada, were similarly polymorphic for 1AT, whereas coyote and red wolf samples were all monomorphic. Our findings, in conjunction with earlier results, are consistent with the Minnesota wolf being a gray wolf of Eurasian origin or possibly a hybrid between the gray wolf of Eurasian origin and the proposed North American wolf.

  19. Fluphenazine Reduces Proteotoxicity in C. elegans and Mammalian Models of Alpha-1-Antitrypsin Deficiency

    PubMed Central

    Li, Jie; Pak, Stephen C.; O’Reilly, Linda P.; Benson, Joshua A.; Wang, Yan; Hidvegi, Tunda; Hale, Pamela; Dippold, Christine; Ewing, Michael; Silverman, Gary A.; Perlmutter, David H.

    2014-01-01

    The classical form of α1-antitrypsin deficiency (ATD) is associated with hepatic fibrosis and hepatocellular carcinoma. It is caused by the proteotoxic effect of a mutant secretory protein that aberrantly accumulates in the endoplasmic reticulum of liver cells. Recently we developed a model of this deficiency in C. Elegans and adapted it for high-content drug screening using an automated, image-based array scanning. Screening of the Library of Pharmacologically Active Compounds identified fluphenazine (Flu) among several other compounds as a drug which reduced intracellular accumulation of mutant α1-antitrypsin Z (ATZ). Because it is representative of the phenothiazine drug class that appears to have autophagy enhancer properties in addition to mood stabilizing activity, and can be relatively easily re-purposed, we further investigated its effects on mutant ATZ. The results indicate that Flu reverses the phenotypic effects of ATZ accumulation in the C. elegans model of ATD at doses which increase the number of autophagosomes in vivo. Furthermore, in nanomolar concentrations, Flu enhances the rate of intracellular degradation of ATZ and reduces the cellular ATZ load in mammalian cell line models. In the PiZ mouse model Flu reduces the accumulation of ATZ in the liver and mediates a decrease in hepatic fibrosis. These results show that Flu can reduce the proteotoxicity of ATZ accumulation in vivo and, because it has been used safely in humans, this drug can be moved rapidly into trials for liver disease due to ATD. The results also provide further validation for drug discovery using C. elegans models that can be adapted to high-content drug screening platforms and used together with mammalian cell line and animal models. PMID:24498058

  20. Hepatic steatosis depresses alpha-1-antitrypsin levels in human and rat acute pancreatitis

    PubMed Central

    Wang, Qian; Du, Jianjun; Yu, Pengfei; Bai, Bin; Zhao, Zhanwei; Wang, Shiqi; Zhu, Junjie; Feng, Quanxin; Gao, Yun; Zhao, Qingchuan; Liu, Chaoxu

    2015-01-01

    Hepatic steatosis (HS) can exacerbate acute pancreatitis (AP). This study aimed to investigate the relation between α1-antitrypsin (AAT) and acute pancreatitis when patients have HS. Using proteomic profiling, we identified 18 differently expressed proteins pots in the serum of rats with or without HS after surgical establishment of AP. AAT was found to be one of the significantly down-regulated proteins. AAT levels were significantly lower in hepatic steatosis acute pancreatitis (HSAP) than in non-HSAP (NHSAP) (P < 0.001). To explore the clinical significance of these observations, we measured the levels of AAT in the serum of 240 patients with HSAP, NHSAP, fatty liver disease (FLD), or no disease. Compared with healthy controls, serum AAT levels in patients with NHSAP were significantly higher (P < 0.01), while in patients with HSAP serum AAT levels were significantly lower (P < 0.01). Further studies showed that acute physiology and chronic health evaluation (APACHE-II) scores were negatively correlated with serum AAT levels (r = −0.85, P < 0.01). In conclusion, low serum levels of AAT in patients with HSAP are correlated with disease severity and AAT may represent a potential target for therapies aiming to improve pancreatitis. PMID:26634430

  1. Mechanistic Evidence in Support of Alpha1-Antitrypsin as a Therapeutic Approach for Type 1 Diabetes

    PubMed Central

    Fleixo-Lima, Gabriella; Ventura, Hilla; Medini, Michal; Bar, Liliana; Strauss, Pnina

    2014-01-01

    Utilizing endogenous molecules as a therapeutic approach is almost unequivocally superior to engineered or synthetic molecules. However, one rarely encounters an anti-inflammatory, cytoprotective, immunomodulatory and wound-healing molecule that has been available for use for decades. α1-antitrypsin (AAT), a circulating protein that rises more than 4-fold during acute-phase responses, has been administered for a rare genetic deficiency at large doses, for life. Aside from advances in insulin therapy, medical research in type 1 diabetes (T1D) has predominantly focused on autoimmunity—controlling the adaptive immune response. However, it is now appreciated that one may need to extend therapeutic targets to incorporate immune responses to cellular injury, as well as promote selective control over excessive inflammation and early tissue repair. Recent data suggest that tissue damage related to lung and renal ischemia-reperfusion injury, stroke, and ischemic heart disease is markedly reduced by AAT. AAT was also shown to protect pancreatic islet β cells at multiple levels. Unlike classic immunosuppressive and anti-inflammatory approaches, AAT exerts some antiviral and antibacterial activities. Based on these and other reports, AAT is under evaluation for treatment of T1D patients in multiple clinical trials. Initial results suggest that AAT therapy could potentially improve insulin production without adverse effects. Up to 50% of individuals displayed improved islet function. It is a rare occurrence in T1D research that a therapy is offered that holds a safety profile equal or superior to that of insulin alone. While placebo-controlled trials are ongoing, the mechanism(s) behind these favorable activities of AAT are still being explored. PMID:25155845

  2. Polymorphism of alpha 1 antitrypsin in North American species of Canis

    USGS Publications Warehouse

    Federoff, N.E.; Kueppers, F.

    2000-01-01

    a1-Antitrypsin (A1AT) is a major protease inhibitor present in all mammalian sera that have thus far been investigated. A1AT is also highly polymorphic and is therefore a useful genetic marker. Previously reported A1AT polymorphism in domestic dogs consisted of two alleles designated as PiM and PiS which exhibited frequencies of 0.72 and 0.28, respectively, in a group of randomly collected mongrel dogs. North American species of Canis, which included gray wolves (n=29), Mexican wolves (n=20), coyotes (n=24), wolfdog crosses (n=9), and red wolves (n=27) were tested for A1AT polymorphism. A1AT phenotypes were determined by isoelectric focusing, followed by direct immunoblotting using a specific antiserum. A1AT concentrations were determined by radial immunodiffusion. Concentrations of A1AT were similar to those found in domestic dogs (2.26 + 0.3 mg/ml SD) and tended to be higher in females than in males, possibly indicating that A1AT may be hormonally influenced in females. Three phenotypic band patterns were observed (M, MS, S). The allele frequencies for domestic dogs and gray wolves were very similar, 0.72 and 0.69 for PiM and 0.28 and 0.31 for PiS, respectively. The Mexican wolves had a significantly lower frequency of PiS= 0.10. Coyotes and red wolves were all found to be monomorphic for the PiS allele and were indistinguishable from each other in that respect.

  3. Oxidative Stress and Polymorphism of Xenobiotic-Metabolizing Enzymes in Two Patients with Severe Alpha-1-Antitrypsin Deficiency.

    PubMed

    Topic, Aleksandra; Nagorni-Obradovic, Ljudmila; Francuski, Djordje; Ljujic, Mila; Malic, Zivka; Radojkovic, Dragica

    2016-10-01

    Alpha-1-antitrypsin deficiency (AATD) and tobacco smoke play a key role in the pathogenesis of early-onset emphysema. Differences in AATD-related chronic obstructive pulmonary disease stages imply the existence of modifying factors associated with disease severity. We present two male patients with emphysema caused by severe AATD (PiZZ genotype). Both are former smokers and have epoxide hydrolase low-activity phenotype. Extremely high level of oxidative stress (high urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine), increased inflammation (high serum CRP), and GSTP1 105Val mutation were found in patient with a worse lung function and prognosis. These data provide more evidence that oxidative stress-related gene variants and inflammation are associated with worse symptoms of AATD-related emphysema. Therefore, prevention against severe stage of AATD-related emphysema would include early identification of the risk gene variants, cessation or never smoking, and treatment with anti-inflammatory and anti-oxidant drugs. Additionally, urinary 8-oxodG could be a candidate for predictive biomarker for routine assessment of the oxidative stress level in AATD patients.

  4. Distribution and levels of alpha-1-antitrypsin in the lung and plasma in smokers and chronic obstructive pulmonary disease.

    PubMed

    Linja-aho, Anna; Mazur, Witold; Toljamo, Tuula; Nieminen, Pentti; Ohlmeier, Steffen; Rönty, Mikko; Kinnula, Vuokko L

    2013-01-01

    Our recent non-biased proteomic screening study revealed elevated SerpinA1 i.e. alpha-1-antitrypsin (AAT) levels in induced sputum of smokers with Chronic obstructive pulmonary disease (COPD). This study was designed to further investigate the role of AAT in smokers and subjects with COPD. The expression/distribution of AAT was studied by immunohistochemistry/digital image morphometry in the lung, by Western blot in the lung and sputum, and by ELISA in the plasma at baseline (n = 349) and after a 2-year follow-up (n = 58). AAT was localized mainly in airway and alveolar epithelium and endothelium, especially in smokers and in those with COPD. AAT was elevated in smokers and in subjects with COPD in the lung endothelial cells. Total lung AAT immunoreactivity was elevated in subjects with moderate COPD compared with smokers and with non-smokers. AAT showed elevated tendency in sputum of smokers with COPD compared with 'healthy' smokers. Plasma AAT levels were elevated in smokers with/without COPD compared with non-smokers. In the follow-up, plasma AAT concentrations decreased significantly after quitting smoking. Chronic smoking/COPD leads to AAT elevation especially in the endothelium of the lung periphery; these changes reflect only modestly to the AAT in sputum, while plasma AAT significantly reflects smoking-related systemic manifestations, and decreases after smoking cessation.

  5. Targeted Biomarker Discovery by High Throughput Glycosylation Profiling of Human Plasma Alpha1-Antitrypsin and Immunoglobulin A

    PubMed Central

    Ruhaak, L. Renee; Koeleman, Carolien A. M.; Uh, Hae-Won; Stam, Jord C.; van Heemst, Diana; Maier, Andrea B.; Houwing-Duistermaat, Jeanine J.; Hensbergen, Paul J.; Slagboom, P. Eline; Deelder, André M.; Wuhrer, Manfred

    2013-01-01

    Protein N-glycosylation patterns are known to show vast genetic as well as physiological and pathological variation and represent a large pool of potential biomarkers. Large-scale studies are needed for the identification and validation of biomarkers, and the analytical techniques required have recently been developed. Such methods have up to now mainly been applied to complex mixtures of glycoproteins in biofluids (e.g. plasma). Here, we analyzed N-glycosylation profiles of alpha1-antitrypsin (AAT) and immunoglobulin A (IgA) enriched fractions by 96-well microtitration plate based high-throughput immuno-affinity capturing and N-glycan analysis using multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). Human plasma samples were from the Leiden Longevity Study comprising 2415 participants of different chronological and biological ages. Glycosylation patterns of AAT enriched fractions were found to be associated with chronological (calendar) age and they differed between females and males. Moreover, several glycans in the AAT enriched fraction were associated with physiological parameters marking cardiovascular and metabolic diseases. Pronounced differences were found between males and females in the glycosylation profiles of IgA enriched fractions. Our results demonstrate that large-scale immuno-affinity capturing of proteins from human plasma using a bead-based method combined with high-throughput N-glycan analysis is a powerful tool for the discovery of glycosylation-based biomarker candidates. PMID:24039863

  6. Novel variants of SERPIN1A gene: Interplay between alpha1-antitrypsin deficiency and chronic obstructive pulmonary disease.

    PubMed

    Bashir, Arif; Shah, Naveed Nazir; Hazari, Younis Mohammad; Habib, Mudasir; Bashir, Samirul; Hilal, Nazia; Banday, Mariam; Asrafuzzaman, Syed; Fazili, Khalid Majid

    2016-08-01

    Alpha1-antitrypsin (AAT) is one of the major circulating anti-protease whose levels in circulation are raised during excessive amount of proteases, especially neutrophil elastase (NE) released during the course of inflammation. Proteolytic attack of NE on peripheral organs, more exclusively on lung parenchyma has severe consequence that may precipitate pulmonary emphysema. Normally, human body has its own molecular and physiological mechanisms to synthesize and regulate the production of anti-protease like AAT to mitigate the extent of inflammatory damage. AAT coded by serine-protease inhibitor (SERPINA1) is predominantly expressed in hepatocytes and to some extent by macrophages, monocytes, lung tissue etc. The observation that persons with AAT deficiency developed chronic obstructive pulmonary disease (COPD) and early-onset of emphysema proposed a role for pathways connecting AAT in pathogenesis. Extensive studies have been done till now to bridge a connection between numerous genetic polymorphisms of SERPINA1 gene and the early onset of COPD. Here in this review, we have comprehensively discussed some of the variants of SERPINA1 gene discovered till date and their association with the exacerbation of obstructive pulmonary disease. PMID:27492524

  7. Congruence-Incongruence Patterns in Alpha-1 Antitrypsin Deficiency Couples' Genetic Determinist Beliefs and Perceived Control over Genes: Implications for Clinical and Public Health Genomic Communication.

    PubMed

    Parrott, Roxanne L; Smith, Rachel A; Hong, Soo Jung; Worthington, Amber

    2015-06-01

    Genomics makes possible the isolation of multiple genes as co-factors that increase, but do not determine, risk for many adult-onset medical conditions, including alpha-1 antitrypsin deficiency (AATD). Those diagnosed with an adult-onset medical condition, such as AATD, are often married and make decisions about testing and care as a couple. We examined genetic essentialist and threat beliefs, focusing on beliefs about the genetic contribution to disease susceptibility and severity, as well as perceptions of control related to genes and health for married couples (N =59), in which one spouse has been tested for genetic mutations associated with AATD. The intraclass correlation for spouses' beliefs about genetic essentialism was strong and statistically significant, but the associations for their other beliefs were not. Incongruence between AATD participants and their spouses regarding genes' influence on disease severity directly related to incongruent perceptions of control and genetic contribution to disease susceptibility. Results revealed an inverse relationship to AATD participants' perceptions of behavioral control and a direct relationship to their beliefs about genes' influence on disease severity. This suggests a pattern of incongruence in which AATD participants have low levels of perceived control over genes' influence on health and high levels of perceived genetic influence on disease severity compared to spouses. With public health communication efforts lagging behind the science of genomics, insights regarding the congruence or incongruence associated with married couples' beliefs about genes' influence on disease afford pathways to guide clinical and public health communication about genomics.

  8. Rationale and Design of the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) Study. Sarcoidosis Protocol

    PubMed Central

    Koth, Laura L.; Maier, Lisa A.; Morris, Alison; Drake, Wonder; Rossman, Milton; Leader, Joseph K.; Collman, Ronald G.; Hamzeh, Nabeel; Sweiss, Nadera J.; Zhang, Yingze; O’Neal, Scott; Senior, Robert M.; Becich, Michael; Hochheiser, Harry S.; Kaminski, Naftali; Wisniewski, Stephen R.; Gibson, Kevin F.

    2015-01-01

    Sarcoidosis is a systemic disease characterized by noncaseating granulomatous inflammation with tremendous clinical heterogeneity and uncertain pathobiology and lacking in clinically useful biomarkers. The Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study is an observational cohort study designed to explore the role of the lung microbiome and genome in these two diseases. This article describes the design and rationale for the GRADS study sarcoidosis protocol. The study addresses the hypothesis that distinct patterns in the lung microbiome are characteristic of sarcoidosis phenotypes and are reflected in changes in systemic inflammatory responses as measured by peripheral blood changes in gene transcription. The goal is to enroll 400 participants, with a minimum of 35 in each of 9 clinical phenotype subgroups prioritized by their clinical relevance to understanding of the pathobiology and clinical heterogeneity of sarcoidosis. Participants with a confirmed diagnosis of sarcoidosis undergo a baseline visit with self-administered questionnaires, chest computed tomography, pulmonary function tests, and blood and urine testing. A research or clinical bronchoscopy with a research bronchoalveolar lavage will be performed to obtain samples for genomic and microbiome analyses. Comparisons will be made by blood genomic analysis and with clinical phenotypic variables. A 6-month follow-up visit is planned to assess each participant’s clinical course. By the use of an integrative approach to the analysis of the microbiome and genome in selected clinical phenotypes, the GRADS study is powerfully positioned to inform and direct studies on the pathobiology of sarcoidosis, identify diagnostic or prognostic biomarkers, and provide novel molecular phenotypes that could lead to improved personalized approaches to therapy for sarcoidosis. PMID:26193069

  9. Rationale and Design of the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) Study. Sarcoidosis Protocol.

    PubMed

    Moller, David R; Koth, Laura L; Maier, Lisa A; Morris, Alison; Drake, Wonder; Rossman, Milton; Leader, Joseph K; Collman, Ronald G; Hamzeh, Nabeel; Sweiss, Nadera J; Zhang, Yingze; O'Neal, Scott; Senior, Robert M; Becich, Michael; Hochheiser, Harry S; Kaminski, Naftali; Wisniewski, Stephen R; Gibson, Kevin F

    2015-10-01

    Sarcoidosis is a systemic disease characterized by noncaseating granulomatous inflammation with tremendous clinical heterogeneity and uncertain pathobiology and lacking in clinically useful biomarkers. The Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study is an observational cohort study designed to explore the role of the lung microbiome and genome in these two diseases. This article describes the design and rationale for the GRADS study sarcoidosis protocol. The study addresses the hypothesis that distinct patterns in the lung microbiome are characteristic of sarcoidosis phenotypes and are reflected in changes in systemic inflammatory responses as measured by peripheral blood changes in gene transcription. The goal is to enroll 400 participants, with a minimum of 35 in each of 9 clinical phenotype subgroups prioritized by their clinical relevance to understanding of the pathobiology and clinical heterogeneity of sarcoidosis. Participants with a confirmed diagnosis of sarcoidosis undergo a baseline visit with self-administered questionnaires, chest computed tomography, pulmonary function tests, and blood and urine testing. A research or clinical bronchoscopy with a research bronchoalveolar lavage will be performed to obtain samples for genomic and microbiome analyses. Comparisons will be made by blood genomic analysis and with clinical phenotypic variables. A 6-month follow-up visit is planned to assess each participant's clinical course. By the use of an integrative approach to the analysis of the microbiome and genome in selected clinical phenotypes, the GRADS study is powerfully positioned to inform and direct studies on the pathobiology of sarcoidosis, identify diagnostic or prognostic biomarkers, and provide novel molecular phenotypes that could lead to improved personalized approaches to therapy for sarcoidosis. PMID:26193069

  10. Corticosteroid-binding globulin cleavage is paradoxically reduced in alpha-1 antitrypsin deficiency: Implications for cortisol homeostasis.

    PubMed

    Nenke, Marni A; Holmes, Mark; Rankin, Wayne; Lewis, John G; Torpy, David J

    2016-01-15

    High-affinity corticosteroid-binding globulin (haCBG) is cleaved by neutrophil elastase (NE) resulting in permanent transition to the low cortisol-binding affinity form (laCBG), thereby increasing cortisol availability at inflammatory sites. Alpha-1 antitrypsin (AAT) is the major inhibitor of NE. AAT deficiency (AATD) predisposes patients to early-onset emphysema due to increased proteolytic destruction from the inherent proteinase-antiproteinase imbalance. We hypothesized that AATD may result in increased CBG cleavage in vivo. We collected demographic data and blood samples from 10 patients with AATD and 28 healthy controls measuring total CBG and haCBG levels by parallel in-house ELISAs, as well as AAT, total and free cortisol levels. haCBG was higher (median [range]); 329 [210-551] vs. 250 [175-365] nmol/L; P<0.005, and laCBG lower; 174 [68-229] vs. 220 [119-348] nmol/L; P=0.016 in the AATD group, compared with controls. The ratio of haCBG:total CBG was also higher in AATD; 72 [53-83] vs. 54 [41-72] %; P=0.0001). There was a negative correlation between haCBG:total CBG and AAT levels (P<0.05, R=-0.64). Paradoxically, proteolytic cleavage of CBG was reduced in AATD, despite the recognized increase in NE activity. This implies that NE activity is not the mechanism for systemic CBG cleavage in basal, low inflammatory conditions. Relatively low levels of laCBG may have implications for cortisol action in AATD.

  11. Ubiquitin ligase gp78 increases solubility and facilitates degradation of the Z variant of {alpha}-1-antitrypsin

    SciTech Connect

    Shen Yuxian; Ballar, Petek; Fang, Shengyun . E-mail: fangs@umbi.umd.edu

    2006-11-03

    Deficiency of circulating {alpha}-1-antitrypsin (AAT) is the most widely recognized abnormality of a proteinase inhibitor that causes lung disease. AAT-deficiency is caused by mutations of the AAT gene that lead to AAT protein retention in the endoplasmic reticulum (ER). Moreover, the mutant AAT accumulated in the ER predisposes the homozygote to severe liver injuries, such as neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. Despite the fact that mutant AAT protein is subject to ER-associated degradation (ERAD), yeast genetic studies have determined that the ubiquitination machinery, Hrd1/Der3p-cue1p-Ubc7/6p, which plays a prominent role in ERAD, is not involved in degradation of mutant AAT. Here we report that gp78, a ubiquitin ligase (E3) pairing with mammalian Ubc7 for ERAD, ubiquitinates and facilitates degradation of ATZ, the classic deficiency variant of AAT having a Z mutation (Glu 342 Lys). Unexpectedly, gp78 over-expression also significantly increases ATZ solubility. p97/VCP, an AAA ATPase essential for retrotranslocation of misfolded proteins from the ER during ERAD, is involved in gp78-mediated degradation of ATZ. Surprisingly, unlike other ERAD substrates that cause ER stress leading to apoptosis when accumulated in the ER, ATZ, in fact, increases cell proliferation when over-expressed in cells. This effect can be partially inhibited by gp78 over-expression. These data indicate that gp78 assumes multiple unique quality control roles over ATZ, including the facilitation of degradation and inhibition of aggregation of ATZ.

  12. Rationale and Design of the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) Study. Sarcoidosis Protocol.

    PubMed

    Moller, David R; Koth, Laura L; Maier, Lisa A; Morris, Alison; Drake, Wonder; Rossman, Milton; Leader, Joseph K; Collman, Ronald G; Hamzeh, Nabeel; Sweiss, Nadera J; Zhang, Yingze; O'Neal, Scott; Senior, Robert M; Becich, Michael; Hochheiser, Harry S; Kaminski, Naftali; Wisniewski, Stephen R; Gibson, Kevin F

    2015-10-01

    Sarcoidosis is a systemic disease characterized by noncaseating granulomatous inflammation with tremendous clinical heterogeneity and uncertain pathobiology and lacking in clinically useful biomarkers. The Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study is an observational cohort study designed to explore the role of the lung microbiome and genome in these two diseases. This article describes the design and rationale for the GRADS study sarcoidosis protocol. The study addresses the hypothesis that distinct patterns in the lung microbiome are characteristic of sarcoidosis phenotypes and are reflected in changes in systemic inflammatory responses as measured by peripheral blood changes in gene transcription. The goal is to enroll 400 participants, with a minimum of 35 in each of 9 clinical phenotype subgroups prioritized by their clinical relevance to understanding of the pathobiology and clinical heterogeneity of sarcoidosis. Participants with a confirmed diagnosis of sarcoidosis undergo a baseline visit with self-administered questionnaires, chest computed tomography, pulmonary function tests, and blood and urine testing. A research or clinical bronchoscopy with a research bronchoalveolar lavage will be performed to obtain samples for genomic and microbiome analyses. Comparisons will be made by blood genomic analysis and with clinical phenotypic variables. A 6-month follow-up visit is planned to assess each participant's clinical course. By the use of an integrative approach to the analysis of the microbiome and genome in selected clinical phenotypes, the GRADS study is powerfully positioned to inform and direct studies on the pathobiology of sarcoidosis, identify diagnostic or prognostic biomarkers, and provide novel molecular phenotypes that could lead to improved personalized approaches to therapy for sarcoidosis.

  13. Heterologous protein expression by transimmortalized differentiated liver cell lines derived from transgenic mice (hepatomas/alpha 1 antitrypsin/ONC mouse).

    PubMed

    Dalemans, W; Perraud, F; Le Meur, M; Gerlinger, P; Courtney, M; Pavirani, A

    1990-07-01

    A number of therapeutic plasma proteins are synthesized by human hepatocytes. Since many of these proteins undergo liver-specific post-translational modifications which are required for full biological activity, it may therefore be necessary to develop hepatocyte-based expression systems for their production. Using transgenic mice we have developed a transimmortalisation technique for the isolation of differentiated hepatic cell lines, already engineered to secrete human alpha 1 antitrypsin (alpha 1 AT), a plasma protein which is produced mainly in liver cells. This was achieved by co-expression of the mouse c-myc proto-oncogene and a genomic copy of the human alpha 1 AT gene, both under the control of the human alpha 1 AT promoter. Transgenic mice carrying this construct developed hepatomas producing human alpha 1 AT. Under defined culture conditions, cell lines secreting active alpha 1 AT were derived from these tumours. These cells maintain a differentiated hepatic phenotype and continue to secrete human alpha 1 AT for at least 40 generations. PMID:2257132

  14. Alpha-1 antitrypsin deficiency

    MedlinePlus

    ... liver from damage. The condition can lead to emphysema and liver disease . ... descent. Adults with severe AAT deficiency will develop emphysema , often before age 40. Smoking can increase the ...

  15. Alpha-1 Antitrypsin Deficiency

    MedlinePlus

    ... the right shape, they get stuck in the liver cells and can't reach the lungs. Symptoms of AAT deficiency include Shortness of breath and wheezing Repeated lung ... or delay lung symptoms. NIH: National Heart, Lung, and Blood Institute

  16. Relevance of classic anti-neutrophil cytoplasmic autoantibody (C-ANCA)-mediated inhibition of proteinase 3-alpha 1-antitrypsin complexation to disease activity in Wegener's granulomatosis.

    PubMed

    Dolman, K M; Stegeman, C A; van de Wiel, B A; Hack, C E; von dem Borne, A E; Kallenberg, C G; Goldschmeding, R

    1993-09-01

    In the sera of patients with Wegener's granulomatosis (WG), C-ANCA can be detected that are directed against proteinase 3 (PR3). We have previously observed that C-ANCA interfere with PR3 proteolytic activity and with complexation of PR3 with its major physiologic inhibitor, alpha 1-antitrypsin (alpha 1AT). In the present study we investigated whether this inhibitory effect of C-ANCA on PR3-alpha 1AT complexation correlates with clinical activity of WG. Serial serum samples of eight consecutive patients with histologically proven relapses of WG were tested. At the moment of relapse all sera revealed inhibitory activity towards PR3-alpha 1AT complexation (median 22%, range 10-59%). Disease activity score (r = 0.87, P < 0.02) and C-reactive protein (CRP) levels (r = 0.66, P < 0.1) correlated with C-ANCA inhibition of PR3-alpha 1AT complexation, while they did not correlate with the C-ANCA titre detected by indirect immunofluorescence (IIF) nor with IgG anti-PR3 antibody level measured by ELISA. The inhibitory effect of C-ANCA on PR3-alpha 1AT complexation had risen significantly at the moment of relapse compared with values 3 months (P < 0.05) and 6 months (P < 0.01) before relapse. Eight patients with established WG and positive for C-ANCA but without clinical evidence of relapse served as controls. In this group no inhibitory effect of C-ANCA on PR3-alpha 1AT complexation was observed in 7/8 patients sera. Sera of one control patient contained moderate C-ANCA inhibitory activity towards PR3-alpha 1AT complexation, which remained at a constant level during the 6 months period of observation. Thus, disease activity in WG appears to be more closely related to C-ANCA inhibitory activity towards PR3-alpha 1AT complexation.

  17. Inhibition of HIV-2(ROD) replication in a lymphoblastoid cell line by the alpha1-antitrypsin Portland variant (alpha1-PDX) and the decRVKRcmk peptide: comparison with HIV-1(LAI).

    PubMed

    Bahbouhi, B; Bendjennat, M; Chiva, C; Kogan, M; Albericio, F; Giralt, E; Seidah, N G; Bahraoui, E

    2001-11-01

    We investigated the effects of alpha1-antitrypsine Portland variant (alpha1-PDX) and decanoylRVKRchloromethylketone (decRVKRcmk) on HIV-2(ROD) replication in the Jurkat lymphoblastoid cell line. To this end, cells were stably transfected with the alpha1-PDX (J-PDX) and used as targets for HIV-2(ROD) infection. Controls were prepared with the empty vector (J-pcDNA3). HIV-2(ROD) and HIV-1(LAI) replications were significantly inhibited and delayed in the presence of the alpha1-PDX protein. When decRVKRcmk was used at 35 microM, inhibition rates were 70-80% for HIV-2(ROD) and HIV-1(LAI), while total inhibition occurred at 70 microM. Control peptides consisting of decanoylRVKR and acetylYVADcmk had no effect. In the presence of the alpha1-PDX or the decRVKRcmk at 35 microM, the infectivity of HIV-2(ROD) and HIV-1(LAI) produced was 3-4-fold lower. Both molecules inhibited syncytium formation by HIV-2(ROD) and HIV-1(LAI) to a considerable extent. Finally, the inhibition of viral replication was correlated with the ability of the decRVKRcmk at 35 and 70 microM and of the alpha1-PDX, to inhibit the processing of envelope glycoprotein precursors. The alpha1-PDX protein and the decRVKRcmk peptide at 35 microM inhibited HIV-2 and HIV-1 to a similar level suggesting that identical or closely related endoproteases are involved in the maturation of their envelope glycoprotein precursors into surface and transmembrane glycoproteins. The significant inhibition observed with alpha1-PDX indicates that furin or furin-like endoproteases appear to play a major role in the maturation process.

  18. Cause-specific mortality in individuals with severe alpha 1-antitrypsin deficiency in comparison with the general population in Sweden

    PubMed Central

    Tanash, Hanan A; Ekström, Magnus; Wagner, Philippe; Piitulainen, Eeva

    2016-01-01

    Background Severe alpha 1-antitrypsin deficiency (PiZZ) predisposes to morbidity and mortality due to early-onset emphysema and liver disease. The risk of death from other causes, including cardiovascular disease and cancer, has not been well investigated. We aimed to analyze cause-specific mortality in PiZZ individuals compared with the general Swedish population. Methods Data on 1,561 PiZZ individuals from the Swedish National AAT Deficiency Register, prospectively followed from 1991 to 2014, were analyzed. Causes of death according to the Swedish National Causes of Death Register for the study group were compared with those for the general Swedish population matched for age, sex, and calendar year, with the excess mortality expressed as standardized mortality ratios (SMRs) with 95% confidence intervals (CIs). Results There were 524 deaths during the follow-up period. PiZZ individuals had excess all-cause mortality compared with the Swedish general population (SMR 3.6, 95% CI 3.3–3.9). SMR for ischemic heart disease (IHD) was 0.5 (95% CI 0.3–0.8) and was similar for never and ever-smokers, and in males and females. SMR for lung cancer was 0.9 (95% CI 0.4–1.7). PiZZ individuals had increased mortality compared with the general population for the following diseases: respiratory disease, SMR 48.4 (95% CI 43.0–54.5); primary liver carcinoma, SMR 90.0 (95% CI 59.3–130.9); complicated colon diverticulitis, SMR 20.8 (95% CI 6.7–48.6); and pulmonary embolism, SMR 6.9 (95% CI 3.3–12.7). Conclusion PiZZ individuals had a reduced mortality risk of IHD. Mortality due to respiratory, hepatic disease, diverticulitis, and pulmonary embolism was markedly increased compared with the age- and sex-matched Swedish population. PMID:27555756

  19. Causal and Synthetic Associations of Variants in the SERPINA Gene Cluster with Alpha1-antitrypsin Serum Levels

    PubMed Central

    Thun, Gian Andri; Kumar, Ashish; Obeidat, Ma'en; Zorzetto, Michele; Haun, Margot; Curjuric, Ivan; Couto Alves, Alexessander; Jackson, Victoria E.; Albrecht, Eva; Ried, Janina S.; Teumer, Alexander; Lopez, Lorna M.; Huffman, Jennifer E.; Enroth, Stefan; Bossé, Yohan; Hao, Ke; Timens, Wim; Gyllensten, Ulf; Polasek, Ozren; Wilson, James F.; Rudan, Igor; Hayward, Caroline; Sandford, Andrew J.; Deary, Ian J.; Koch, Beate; Reischl, Eva; Schulz, Holger; Hui, Jennie; James, Alan L.; Rochat, Thierry; Russi, Erich W.; Jarvelin, Marjo-Riitta; Strachan, David P.; Hall, Ian P.; Tobin, Martin D.; Dahl, Morten; Fallgaard Nielsen, Sune; Nordestgaard, Børge G.; Kronenberg, Florian; Luisetti, Maurizio; Probst-Hensch, Nicole M.

    2013-01-01

    Several infrequent genetic polymorphisms in the SERPINA1 gene are known to substantially reduce concentration of alpha1-antitrypsin (AAT) in the blood. Since low AAT serum levels fail to protect pulmonary tissue from enzymatic degradation, these polymorphisms also increase the risk for early onset chronic obstructive pulmonary disease (COPD). The role of more common SERPINA1 single nucleotide polymorphisms (SNPs) in respiratory health remains poorly understood. We present here an agnostic investigation of genetic determinants of circulating AAT levels in a general population sample by performing a genome-wide association study (GWAS) in 1392 individuals of the SAPALDIA cohort. Five common SNPs, defined by showing minor allele frequencies (MAFs) >5%, reached genome-wide significance, all located in the SERPINA gene cluster at 14q32.13. The top-ranking genotyped SNP rs4905179 was associated with an estimated effect of β = −0.068 g/L per minor allele (P = 1.20*10−12). But denser SERPINA1 locus genotyping in 5569 participants with subsequent stepwise conditional analysis, as well as exon-sequencing in a subsample (N = 410), suggested that AAT serum level is causally determined at this locus by rare (MAF<1%) and low-frequent (MAF 1–5%) variants only, in particular by the well-documented protein inhibitor S and Z (PI S, PI Z) variants. Replication of the association of rs4905179 with AAT serum levels in the Copenhagen City Heart Study (N = 8273) was successful (P<0.0001), as was the replication of its synthetic nature (the effect disappeared after adjusting for PI S and Z, P = 0.57). Extending the analysis to lung function revealed a more complex situation. Only in individuals with severely compromised pulmonary health (N = 397), associations of common SNPs at this locus with lung function were driven by rarer PI S or Z variants. Overall, our meta-analysis of lung function in ever-smokers does not support a functional role of common SNPs in

  20. PCR-based screening for the most prevalent alpha 1 antitrypsin deficiency mutations (PI S, Z, and Mmalton) in COPD patients from Eastern Tunisia.

    PubMed

    Denden, Sabri; Lakhdar, Ramzi; Keskes, Nadia Boudawara; Hamdaoui, Mohamed Hedi; Chibani, Jemni Ben; Khelil, Amel Haj

    2013-10-01

    It is generally agreed that the protease inhibitor (PI) alleles PI*S (Val264Glu) and PI*Z (Lys342Glu) are the most common alpha 1 antitrypsin deficiency variants worldwide, but the PI*Mmalton allele (ΔPhe52) prevails over these variants in some Mediterranean regions. In eastern Tunisia (Mahdia), we screened 100 subjects with chronic obstructive pulmonary disease for these variants. The PI*S and PI*Z alleles were genotyped by the previously described SexAI/Hpγ99I RFLP-PCR. We provide here a new method for PI*Mmalton genotyping using mismatched RFLP-PCR. These methods are suitable for routine clinical application and can easily be reproduced by several laboratories, since they do not require extensive optimization, unlike the previously described bidirectional allele-specific amplification PCR for PI*Mmalton genotyping. Our results were in agreement with previous reports from central Tunisia (Kairouan), suggesting that the PI*Mmalton mutation is the most frequent alpha 1 antitrypsin deficiency-related mutation in Tunisia.

  1. Met 358 to Arg mutation of alpha 1-antitrypsin associated with protein C deficiency in a patient with mild bleeding tendency.

    PubMed Central

    Vidaud, D; Emmerich, J; Alhenc-Gelas, M; Yvart, J; Fiessinger, J N; Aiach, M

    1992-01-01

    The molecular defect responsible for a dramatic prolongation of all standard clotting tests discovered in a 15-yr-old boy has been identified. Initial investigations revealed the presence of an activated Factor X (Factor Xa) and thrombin inhibitor which copurified with alpha 1-antitrypsin (alpha 1-AT), thereby suggesting the occurrence of an alpha 1-AT variant similar to alpha 1-AT Pittsburgh. This was confirmed by dot-blot analysis and direct sequencing after amplification by the polymerase chain reaction. A G to T transition at nucleotide 10038 results in the substitution of Met to an Arg, converting alpha 1-AT into an Arg-Ser protease inhibitor (serpin) that inhibited thrombin and Factor Xa more effectively than antithrombin III. Surprisingly, there was no bleeding history in the proband. The common mutation Z, which may explain a reduced expression of the allele bearing the Arg 358 Met mutation, was not observed in the propositus' DNA. To exclude the presence of another mutation, the coding regions and intron/exon junctions were sequenced. No other mutation was found. Recently, the patient experienced his first hemorrhagic episode at the age of 17. The level of the abnormal inhibitor had increased twofold 2 mo before. The large decrease in protein C concentration may account for the mild bleeding tendency in this case, despite the presence of the alpha 1-AT Pittsburgh mutation. An abnormal protein C pattern was observed in patient's plasma, suggesting that the circulating deficiency might be due to a deleterious effect of the abnormal inhibitor on both intracellular processing and catabolism of protein C. Images PMID:1569192

  2. A study of common Mendelian disease carriers across ageing British cohorts: meta-analyses reveal heterozygosity for alpha 1-antitrypsin deficiency increases respiratory capacity and height

    PubMed Central

    North, Teri-Louise; Ben-Shlomo, Yoav; Cooper, Cyrus; Deary, Ian J; Gallacher, John; Kivimaki, Mika; Kumari, Meena; Martin, Richard M; Pattie, Alison; Sayer, Avan Aihie; Starr, John M; Wong, Andrew; Kuh, Diana; Rodriguez, Santiago; Day, Ian N M

    2016-01-01

    Background Several recessive Mendelian disorders are common in Europeans, including cystic fibrosis (CFTR), medium-chain-acyl-Co-A-dehydrogenase deficiency (ACADM), phenylketonuria (PAH) and alpha 1-antitrypsin deficiency (SERPINA1). Methods In a multicohort study of >19 000 older individuals, we investigated the relevant phenotypes in heterozygotes for these genes: lung function (forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC)) for CFTR and SERPINA1; cognitive measures for ACADM and PAH; and physical capability for ACADM, PAH and SERPINA1. Results Findings were mostly negative but lung function in SERPINA1 (protease inhibitor (PI) Z allele, rs28929474) showed enhanced FEV1 and FVC (0.13 z-score increase in FEV1 (p=1.7×10−5) and 0.16 z-score increase in FVC (p=5.2×10−8)) in PI-MZ individuals. Height adjustment (a known, strong correlate of FEV1 and FVC) revealed strong positive height associations of the Z allele (1.50 cm increase in height (p=3.6×10−10)). Conclusions The PI-MZ rare (2%) SNP effect is nearly four times greater than the ‘top’ common height SNP in HMGA2. However, height only partially attenuates the SERPINA1-FEV1 or FVC association (around 50%) and vice versa. Height SNP variants have recently been shown to be positively selected collectively in North versus South Europeans, while the Z allele high frequency is localised to North Europe. Although PI-ZZ is clinically disadvantageous to lung function, PI-MZ increases both height and respiratory function; potentially a balanced polymorphism. Partial blockade of PI could conceivably form part of a future poly-therapeutic approach in very short children. The notion that elastase inhibition should benefit patients with chronic obstructive pulmonary disease may also merit re-evaluation. PI is already a therapeutic target: our findings invite a reconsideration of the optimum level in respiratory care and novel pathway potential for development of agents for the

  3. Classifying Married Adults Diagnosed with Alpha-1 Antitrypsin Deficiency Based on Spousal Communication Patterns Using Latent Class Analysis: Insights for Intervention

    PubMed Central

    Smith, Rachel A.; Wienke, Sara E.; Baker, Michelle K.

    2013-01-01

    Married adults are increasingly exposed to test results that indicate an increased genetic risk for adult-onset conditions. For example, a SERPINA1 mutation, associated with alpha-1 antitrypsin deficiency (AATD), predisposes affected individuals to diseases such as chronic obstructive pulmonary disease (COPD) and cancer, which are often detected in adulthood. Married adults are likely to discuss genetic test results with their spouses, and interpersonal research suggests that spouses’ communication patterns differ. Latent class analysis was used to identify subgroups of spousal communication patterns about AATD results from a sample of married adults in the Alpha-1 Research Registry (N = 130). A five-class model was identified, and the subgroups were consistent with existing spousal-communication typologies. This study also showed that genetic beliefs (e.g., genetic stigma), emotions, and experiences (e.g., insurance difficulties) covaried with membership in particular subgroups. Understanding these differences can serve as the foundation for the creation of effective, targeted communications interventions to address the specific needs and conversational patterns of different kinds of couples. PMID:24177906

  4. Inflammatory cytokine response to exercise in alpha-1-antitrypsin deficient COPD patients ‘on’ or ‘off’ augmentation therapy

    PubMed Central

    2014-01-01

    Background There is still limited information on systemic inflammation in alpha-1-antitrypsin-deficient (AATD) COPD patients and what effect alpha-1-antitrypsin augmentation therapy and/or exercise might have on circulating inflammatory cytokines. We hypothesized that AATD COPD patients on augmentation therapy (AATD + AUG) would have lower circulating and skeletal muscle inflammatory cytokines compared to AATD COPD patients not receiving augmentation therapy (AATD-AUG) and/or the typical non-AATD (COPD) patient. We also hypothesized that cytokine response to exercise would be lower in AATD + AUG compared to AATD-AUG or COPD subjects. Methods Arterial and femoral venous concentration and skeletal muscle expression of TNFα, IL-6, IL-1β and CRP were measured at rest, during and up to 4-hours after 50% maximal 1-hour knee extensor exercise in all COPD patient groups, including 2 additional groups (i.e. AATD with normal lung function, and healthy age-/activity-matched controls). Results Circulating CRP was higher in AATD + AUG (4.7 ± 1.6 mg/dL) and AATD-AUG (3.3 ± 1.2 mg/dL) compared to healthy controls (1.5 ± 0.3 mg/dL, p < 0.05), but lower in AATD compared to non-AATD-COPD patients (6.1 ± 2.6 mg/dL, p < 0.05). TNFα, IL-6 and IL-1β were significantly increased by 1.7-, 1.7-, and 4.7-fold, respectively, in non-AATD COPD compared to AATD COPD (p < 0.05), and 1.3-, 1.7-, and 2.2-fold, respectively, compared to healthy subjects (p < 0.05). Skeletal muscle TNFα was on average 3–4 fold greater in AATD-AUG compared to the other groups (p < 0.05). Exercise showed no effect on these cytokines in any of our patient groups. Conclusion These data show that AATD COPD patients do not experience the same chronic systemic inflammation and exhibit reduced inflammation compared to non-AATD COPD patients. Augmentation therapy may help to improve muscle efflux of TNFα and reduce muscle TNFα concentration, but showed no

  5. Well-Known and Less Well-Known Functions of Alpha-1 Antitrypsin. Its Role in Chronic Obstructive Pulmonary Disease and Other Disease Developments.

    PubMed

    Janciauskiene, Sabina; Welte, Tobias

    2016-08-01

    Alpha-1 antitrypsin (A1AT) is an acute-phase protein, and is best known as an inhibitor of the serine proteases, specifically, neutrophil elastase, proteinase 3, and cathepsin G. The discovery of the connection between inherited A1AT deficiency and emphysema resulted in the concept of a proteinase-antiproteinase imbalance to explain the pathogenic mechanisms of chronic obstructive pulmonary disease, as well as the concomitant development of augmentation therapy with plasma-purified human A1AT. This proteinase-antiproteinase imbalance concept has been difficult to prove, as no single mechanism can account for the complex pathology of chronic obstructive pulmonary disease. New studies have begun to characterize A1AT as an antiinflammatory and an immunoregulatory protein, independent of its antiprotease activity. We recently found that A1AT binds to free fatty acids, and it is this form of A1AT that induces the expression and release of angiopoietin-like protein 4, a protein associated with dyslipidemia and inflammation. This latter finding further strengthens the idea that describing A1AT therapy as antiserine protease is perhaps an oversimplification. The preliminary findings suggest that A1AT could be used for the management of diseases not necessarily related to inherited A1ATD, and points toward a need for more detailed investigations into the relationships between the concentration, structure, and function of A1AT protein. PMID:27564662

  6. Validation and development of an immunonephelometric assay for the determination of alpha-1 antitrypsin levels in dried blood spots from patients with COPD*

    PubMed Central

    Zillmer, Laura Russo; Russo, Rodrigo; Manzano, Beatriz Martins; Ivanaga, Ivan; Nascimento, Oliver Augusto; de Souza, Altay Alves Lino; Santos, Gildo; Rodriguez, Francisco; Miravitlles, Marc; Jardim, José Roberto

    2013-01-01

    OBJECTIVE: To validate and develop an immunonephelometric assay for the determination of alpha-1 antitrypsin (AAT) levels in dried blood spots from COPD patients in Brazil. METHODS: We determined AAT levels in serum samples and dried blood spots from 192 COPD patients. For the preparation of dried blood spots, a disk (diameter, 6 mm) was placed into a tube, eluted with 200 µL of PBS, and stored overnight at 4ºC. All of the samples were analyzed by immunonephelometry in duplicate. We used the bootstrap resampling method in order to determine a cut-off point for AAT levels in dried blood spots. RESULTS: The correlation coefficient between the AAT levels in serum samples and those in dried blood spots was r = 0.45. For dried blood spots, the cut-off value was 2.02 mg/dL (97% CI: 1.45-2.64 mg/dL), with a sensitivity, specificity, positive predictive value, and negative predictive value of 100%, 95.7%, 27.2%, and 100%, respectively. CONCLUSIONS: This method for the determination of AAT levels in dried blood spots appears to be a reliable screening tool for patients with AAT deficiency. PMID:24310627

  7. The role and importance of glycosylation of acute phase proteins with focus on alpha-1 antitrypsin in acute and chronic inflammatory conditions.

    PubMed

    McCarthy, Cormac; Saldova, Radka; Wormald, Mark R; Rudd, Pauline M; McElvaney, Noel G; Reeves, Emer P

    2014-07-01

    Acute phase proteins (APPs) are a group of circulating plasma proteins which undergo changes quantitatively or qualitatively at the time of inflammation. Many of these APPs are glycosylated, and it has been shown that alterations in glycosylation may occur in inflammatory and malignant conditions. Changes in glycosylation have been studied as potential biomarkers in cancer and also in chronic inflammatory conditions and have been shown to correlate with disease severity in certain conditions. Serine protease inhibitors (serpins), many of which are also APPs, are proteins involved in the control of proteases in numerous pathways. Alpha-1 Antitrypsin (AAT) is the most abundant serpin within the circulation and is an APP which has been shown to increase in response to inflammation. The primary role of AAT is maintaining the protease/antiprotease balance in the lung, but it also possesses important anti-inflammatory and immune-modulating properties. Several glycoforms of AAT exist, and they possess differing properties in regard to plasma half-life and stability. Glycosylation may also be important in determining the immune modulatory properties of AAT. The review will focus on the role and importance of glycosylation in acute phase proteins with particular attention to AAT and its use as a biomarker of disease. The review describes the processes involved in glycosylation, how glycosylation changes in differing disease states, and the alterations that occur to glycans of APPs with disease and inflammation. Finally, the review explores the importance of changes in glycosylation of AAT at times of inflammation and in malignant conditions and how this may impact upon the functions of AAT.

  8. The impact of smoke exposure on the clinical phenotype of alpha-1 antitrypsin deficiency in Ireland: exploiting a national registry to understand a rare disease.

    PubMed

    O'Brien, M Emmet; Pennycooke, Kevin; Carroll, Tomás P; Shum, Jonathan; Fee, Laura T; O'Connor, Catherine; Logan, P Mark; Reeves, Emer P; McElvaney, Noel G

    2015-05-01

    Individuals with Alpha-1 antitrypsin deficiency (AATD) have mutations in the SERPINA1 gene causing genetic susceptibility to early onset lung and liver disease that may result in premature death. Environmental interactions have a significant impact in determining the disease phenotype and outcome in AATD. The aim of this study was to assess the impact of smoke exposure on the clinical phenotype of AATD in Ireland. Clinical demographics and available thoracic computerised tomography (CT) imaging were detected from 139 PiZZ individuals identified from the Irish National AATD Registry. Clinical information was collected by questionnaire. Data was analysed to assess AATD disease severity and evaluate predictors of clinical phenotype. Questionnaires were collected from 107/139 (77%) and thoracic CT evaluation was available in 72/107 (67.2%). 74% of respondents had severe Chronic Obstructive Pulmonary Disease (COPD) (GOLD stage C or D). Cigarette smoking was the greatest predictor of impairment in FEV1 and DLCO (%predicted) and the extent of emphysema correlated most significantly with DLCO. Interestingly the rate of FEV1 decline was similar in ex-smokers when compared to never-smokers. Passive smoke exposure in childhood resulted in a greater total pack-year smoking history. Radiological evidence of bronchiectasis was a common finding and associated with increasing age. The Irish National AATD Registry facilitates clinical and basic science research of this condition in Ireland. This study illustrates the detrimental effect of smoke exposure on the clinical phenotype of AATD in Ireland and the benefit of immediate smoking cessation at any stage of lung disease.

  9. Altered glycosylation, expression of serum haptoglobin and alpha-1-antitrypsin in chronic hepatitis C, hepatitis C induced liver cirrhosis and hepatocellular carcinoma patients.

    PubMed

    Mondal, Gautam; Saroha, Ashish; Bose, Partha Pratim; Chatterjee, B P

    2016-04-01

    Liver cirrhosis with hepatitis C viral infection (HCV-LC) causes high risk to develop hepatocellular carcinoma (HCC). Besides diagnosis of liver cirrhosis by biochemical test, imaging techniques, assessment of structural liver damage by biopsy shows several disadvantages. Our aim was to monitor the changes in the expression level of serum proteins and their glycosylation pattern among chronic hepatitis C (HCV-CH), HCV-LC and HCC patients with respect to controls. 2D gel electrophoresis of HCV-CH, HCV-LC and HCC patients' sera showed several protein spots, which were identified by LC-MS. The change in the expression of two prominent protein spots, haptoglobin (Hp) and alpha 1-antitrypsin (AAT) was evaluated by western blot and ELISA. The changes in glycosylation pattern of these serum proteins were assayed using different lectins. Increased level of Hp and AAT was observed in HCV-LC and HCC patients' group whereas those were found to be present less in HCV-CH patient groups with respect to control as determined by ELISA using monoclonal antibodies. Decreased level of sialylation in both Hp and AAT was observed in HCV-LC and HCV-CH patients' group whereas increased level of sialylation was observed in HCC patient groups by ELISA using Sambucus nigra agglutinin. On the other hand increased level of fucosylation in two serum glycoproteins was observed in HCV-LC and HCC patients' group using Lens culinarris agglutinin. High glycan branching was found in HCV-LC and HCC patient groups in Hp but not in HCV-CH as determined by Datura stramonium agglutinin. However, there was no such change observed in glycan branching in AAT of HCV-CH and HCV-LC patients' groups, to the contrary high glycan branching was observed in HCC patients' group. Increased level of exposed galactose in both serum proteins was observed in both HCC patients' group as determined by Ricinus communis agglutinin. The present glycoproteomics study could predict the progression of HCV-CH, HCV-LC and HCC

  10. Usefulness of the CAT, LCOPD, EQ-5D and COPDSS scales in understanding the impact of lung disease in patients with alpha-1 antitrypsin deficiency.

    PubMed

    Manca, Sandra; Rodriguez, Esther; Huerta, Arturo; Torres, Maria; Lazaro, Lourdes; Curi, Sergio; Pirina, Pietro; Miravitlles, Marc

    2014-09-01

    Alpha-1-antitrypsin deficiency (AATD) is an inherited disorder responsible for early onset emphysema associated with a significant impairment of health-related quality of life (HRQoL). Our aim was to assess the usefulness of different instruments to evaluate the HRQoL in patients with AATD compared to non-AATD COPD. Observational, cross-sectional study in which all patients filled out a series of questionnaires: the COPD severity score (COPDSS), the EuroQoL 5-Dimensions (EQ-5D), the Living with COPD (LCOPD) and the COPD Assessment Test (CAT). A total of 96 patients were included, 35 with AATD (mean age 56.5 yrs, 57.1% male and mean FEV1(%) 48.7% and 61 non-AATD COPD (70.3 yrs, 80.3% men and FEV1(%) 47%. The questionnaire scores were similar, with a tendency towards worse scores in AATD for the EQ-5D (VAS) (64.8 (20.2) vs. 71.6 (17.1); p = 0.08). The correlations between the different scores and FEV1(%) were significant in both groups for COPDSS and LCOPD, but not for CAT and EQ-5D. In general, the correlations of scores with FEV1(%) were stronger for AATD compared with non-AATD COPD patients: COPDSS r = -0.570, p < 0.01 for AATD and r = -0.260, p < 0.05 for COPD; LCOPD r = -0.502, p < 0.001 for AATD and r = -0.304, p < 0.05 for non-AATD COPD. Patients with AATD have a similar degree of HRQoL impairment as older subjects with non-AATD COPD and showed a stronger correlation between HRQoL measurements and lung function impairment compared with non-AATD COPD. This may be related to the characteristics of the disease in these patients who are usually younger, with less co-morbidity and lower smoking consumption.

  11. Low Serum Levels of Alpha1 Anti-trypsin (α1-AT) and Risk of Airflow Obstruction in Non-Primary α1-AT-Deficient Patients with Compensated Chronic Liver Disease

    PubMed Central

    Rodríguez-Romero, Elizabeth; Suárez-Cuenca, Juan Antonio; Elizalde-Barrera, César Iván; Mondragón-Terán, Paul; Martínez-Hernández, José Enrique; Gómez-Cortés, Eduardo; de Vaca, Rebeca Pérez-Cabeza; Hernández-Muñoz, Rolando E.; Melchor-López, Alberto; Jiménez-Saab, Nayeli Gabriela

    2015-01-01

    Background Alpha1 anti-trypsin (α1-AT), a serine protease inhibitor synthesized in the liver, is a major circulating antiprotease that provides defense against proteolytic damage in several tissues. Its deficiency is associated with airflow obstruction. The present study aimed to explore the role of α1-AT as a biomarker of airflow performance in chronic liver disease (CLD). Material/Methods Serum α1-AT levels and lung function (spirometry) were evaluated in non-primary α1-AT-deficient, alcoholic CLD patients without evident respiratory limitations. Results Thirty-four patients with airflow obstruction (n=11), airflow restriction (n=12), and normal airflow (n=11, age-matched controls) were eligible. α1-AT was decreased in the airflow obstruction group. ROC-cutoff α1-AT=24 mg/dL effectively discriminated airflow obstruction (AUC=0.687) and was associated with a 10-fold higher risk (p=0.0007). Conclusions Lower α1-AT increased the risk of airflow obstruction in CLD patients without primary α1-AT deficiency. PMID:25913248

  12. HIV Replication in CD4+ T Lymphocytes in the Presence and Absence of Follicular Dendritic Cells: Inhibition of Replication mediated by Alpha-1-Antitrypsin through Altered IκBα Ubiquitination1

    PubMed Central

    Zhou, Xueyuan; Shapiro, Leland; Fellingham, Gilbert; Willardson, Barry M.; Burton, Gregory F.

    2011-01-01

    Follicular dendritic cells (FDCs) increase HIV replication and virus production in lymphocytes by increasing the activation of NF-κB in infected cells. Because alpha-1-antitrypsin (AAT) decreases HIV replication in PBMCs and monocytic cells and decreases NF-κB activity, we postulated that AAT might also block FDC-mediated HIV replication. Primary CD4+ T cells were infected with HIV and cultured with FDCs or their supernatant with or without AAT, and ensuing viral RNA and p24 production were monitored. NF-κB activation in the infected cells was also assessed. Virus production was increased in the presence of FDC supernatant but the addition of AAT at concentrations above 0.5 mg/ml inhibited virus replication. AAT blocked the nuclear translocation of NF-κB p50/p65 despite an unexpected elevation in associated phosphorylated and ubiquitinated IκBα (Ub-IκBα). In the presence of AAT, degradation of cytoplasmic IκBα was dramatically inhibited compared to control cultures. AAT did not inhibit the proteasome; however, it altered the pattern of ubiquitination of IκBα. AAT decreased IκBα polyubiquitination linked through ubiquitin lysine residue 48 (K48) and increased ubiquitination linked through lysine residue 63 (K63). Moreover, K63 linked Ub-IκBα degradation was substantially slower than K48 linked Ub-IκBα in the presence of AAT, correlating altered ubiquitination with a prolonged IκBα half-life. Because AAT is naturally occurring and is available clinically, examination of its use as an inhibitory agent in HIV-infected subjects may be informative and lead to the development of similar agents that inhibit HIV replication using a novel mechanism. PMID:21263074

  13. Living with Alpha-1 Antitrypsin Deficiency

    MedlinePlus

    ... Support Living with AAT deficiency may cause fear, anxiety, depression, and stress. Talk about how you feel ... and friends also can help relieve stress and anxiety. Let your loved ones know how you feel ...

  14. Astute, Assertive, and Alpha-1: Quantifying Empowerment in a Rare Genetic Community

    ERIC Educational Resources Information Center

    Finn, Symma

    2008-01-01

    We investigated empowerment in the Alpha-1 Antitrypsin Deficiency (Alpha-1) community, a rare, genetic disease network in the United States. The research was motivated by nine years of observations in the community. After observing what seemed to be a heightened amount of activism among Alpha-1 community members, I had hypothesized that this…

  15. Biosynthesis and secretion of M- and Z-type alpha 1-proteinase inhibitor by human monocytes. Effect of inhibitors of glycosylation and of oligosaccharide processing on secretion and function.

    PubMed

    Gross, V; vom Berg, D; Kreuzkamp, J; Ganter, U; Bauer, J; Würtemberger, G; Schulz-Huotari, C; Beeser, H; Gerok, W

    1990-03-01

    The biosynthesis and secretion of M-type and Z-type alpha 1-antitrypsin was studied in human monocytes. In monocytes of PiMM individuals alpha 1-antitrypsin represented 0.08% of the newly synthesized proteins and 0.44% of the secreted proteins. Two molecular forms of alpha 1-antitrypsin could be identified: a 51-kDa intracellular form, susceptible to endoglucosaminidase H, thus representing the high-mannose type precursor form and a 56-kDa form resistant to endoglucosaminidase H which was secreted into the medium. Inhibition of de novo glycosylation by tunicamycin impaired the secretion of M-type alpha 1-antitrypsin by about 75% whereas inhibition of oligosaccharide processing by the mannosidase II inhibitor swainsonine did not alter the secretion of M-type alpha 1-antitrypsin. alpha 1-Antitrypsin secreted by human monocytes was functionally active as measured by complex formation with porcine pancreatic elastase. Even unglycosylated alpha 1-antitrypsin secreted by human monocytes treated with tunicamycin formed a complex with elastase. In monocytes of PiZZ individuals the secretion of alpha 1-antitrypsin was decreased. 72% of newly synthesized M-type alpha 1-antitrypsin, but only 35% of newly synthesized Z-type alpha 1-antitrypsin were secreted during a labeling period of 3 h with [35S]methionine. The 51-kDa form of Z-type alpha 1-antitrypsin accumulated intracellularly, whereas the 56-kDa form was secreted. Inhibition of oligosaccharide processing by swainsonine did not alter the decreased secretion of Z-type alpha 1-antitrypsin, whereas inhibition of de novo glycosylation by tunicamycin blocked the secretion of Z-type alpha 1-antitrypsin completely. PMID:2111144

  16. α1-Antitrypsin Deficiency.

    PubMed

    Hatipoğlu, Umur; Stoller, James K

    2016-09-01

    α1-Antitrypsin deficiency is an autosomal codominant condition that predisposes to emphysema and cirrhosis. The condition is common but grossly under-recognized. Identifying patients' α1-antitrypsin deficiency has important management implications (ie, smoking cessation, genetic and occupational counseling, and specific treatment with the infusion of pooled human plasma α1-antitrypsin). The weight of evidence suggests that augmentation therapy slows the progression of emphysema in individuals with severe α1-antitrypsin deficiency. PMID:27514595

  17. Effect of cigarette smoke on human serum trypsin inhibitory capacity and antitrypsin concentration

    SciTech Connect

    Chowdhury, P.; Bone, R.C.; Louria, D.B.; Rayford, P.L.

    1982-07-01

    Investigation of the effect of cigarette smoke on the serum trypsin inhibitory capacity (TIC) and antitrypsin content in 89 smokers compared with 37 nonsmokers revealed that cigarette smoking is associated with a significantly lower level of TIC. No alteration in serum antitrypsin content was found because of cigarette smoking. Further analysis of the data indicated a correlation between the magnitude of smoking and the reduction in serum TIC. The reduction of TIC in cigarette smokers is consistent with the recent findings of decreased alpha 1-antitrypsin activity in rat lung and the reduced elastase inhibitory capacity per mg of alpha 1-antitrypsin found in the serum of smokers. The decrease in TIC in the serum of smokers, in addition to the reported decrease in elastolytic activity, may be useful in explaining the pathogenesis of emphysema frequently found in smokers.

  18. Hypersensitivity Vasculitis with Leukocytoclastic Vasculitis Associated with Alpha-1-Proteinase Inhibitor

    PubMed Central

    Mwirigi, Nicola W.; Thomas, Charles F.

    2009-01-01

    Prolastin is a commercially available form of alpha-1-antitrypsin (AAT) that is derived from pooled human plasma and used for treatment of severe alpha-1-antitrypsin deficiency (AATD). We describe a patient with AATD who developed presumed hypersensitivity vasculitis (HV) following a Prolastin infusion. Hypersensitivity vasculitis (HV), or cutaneous vasculitis, is characterized by inflammation of the small vessels of the skin with resultant ischemia to the distally supplied areas. To our knowledge, this is the first reported case of presumed hypersensitivity vasculitis following Prolastin infusion. PMID:20204065

  19. α1-Antitrypsin deficiency.

    PubMed

    Greene, Catherine M; Marciniak, Stefan J; Teckman, Jeffrey; Ferrarotti, Ilaria; Brantly, Mark L; Lomas, David A; Stoller, James K; McElvaney, Noel G

    2016-01-01

    α1-Antitrypsin deficiency (A1ATD) is an inherited disorder caused by mutations in SERPINA1, leading to liver and lung disease. It is not a rare disorder but frequently goes underdiagnosed or misdiagnosed as asthma, chronic obstructive pulmonary disease (COPD) or cryptogenic liver disease. The most frequent disease-associated mutations include the S allele and the Z allele of SERPINA1, which lead to the accumulation of misfolded α1-antitrypsin in hepatocytes, endoplasmic reticulum stress, low circulating levels of α1-antitrypsin and liver disease. Currently, there is no cure for severe liver disease and the only management option is liver transplantation when liver failure is life-threatening. A1ATD-associated lung disease predominately occurs in adults and is caused principally by inadequate protease inhibition. Treatment of A1ATD-associated lung disease includes standard therapies that are also used for the treatment of COPD, in addition to the use of augmentation therapy (that is, infusions of human plasma-derived, purified α1-antitrypsin). New therapies that target the misfolded α1-antitrypsin or attempt to correct the underlying genetic mutation are currently under development. PMID:27465791

  20. [α1-Antitrypsin deficiency].

    PubMed

    Hirai, Toyohiro

    2016-05-01

    α1-Antitrypsin deficiency (AATD) is the commonest genetic risk factor for developing chronic obstructive pulmonary disease. In 2015, AATD has been categorized as one of intractable diseases called "Nanbyo" in Japan. The prevalence of AATD is extremely low in Japanese compared with Caucasians in North America and Europe. According to recent nationwide epidemiological survey, the prevalence of AATD in Japan was estimated to be 24 patients with a 95% confidence interval. The mutation PI*S(iiyama) is commonly found in the Japanese patients with AATD, whereas PI*Z is the most frequent mutation associated with severe deficiency in Caucasians. The availability of AAT augmentation therapy in Japan is expected. This paper reviews the diagnosis and treatment in AATD. PMID:27254961

  1. Streptomyces erythraeus Trypsin Inactivates α1-Antitrypsin

    PubMed Central

    Vukoti, Krishna M.; Kadiyala, Chandra Sekhar Rao; Miyagi, Masaru

    2011-01-01

    Streptomyces erythraeus trypsin (SET) is a serine protease that is secreted extracellularly by Streptomyces erythraeus. We investigated the inhibitory effect of α1-antitrypsin on the catalytic activity of SET. Intriguingly, we found that SET is not inhibited by α1-antitrypsin. Our investigations into the molecular mechanism underlying this observation revealed that SET hydrolyzes the Met-Ser bond in the reaction center loop of α1-antitrypsin. However, SET somehow avoids entrapment by α1-antitrypsin. We also confirmed that α1-antitrypsin loses its inhibitory activity after incubation with SET. Thus, our study demonstrates that SET is not only resistant to α1-antitrypsin but also inactivates α1-antitrypsin. PMID:22115549

  2. Alpha-1 antitrypsin gene therapy prevented bone loss in ovariectomy induced osteoporosis mouse model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Osteoporosis is a major healthcare burden affecting mostly postmenopausal women characterized by compromised bone strength and increased risk of fragility fracture. Although pathogenesis of this disease is complex, elevated proinflammatory cytokine production is clearly involved in bone loss at meno...

  3. Baseline Analysis of a Young Alpha-1-AT Deficiency Liver Disease Cohort Reveals Frequent Portal Hypertension

    PubMed Central

    Teckman, Jeffrey H; Rosenthal, Philip; Abel, Robert; Bass, Lee M.; Michail, Sonia; Murray, Karen F.; Rudnick, David A.; Thomas, Daniel W.; Spino, Cathie; Arnon, Ronen; Hertel, Paula M.; Heubi, James; Kamath, Binita M.; Karnsakul, Wikrom; Loomes, Kathleen M.; Magee, John C.; Molleston, Jean P.; Romero, Rene; Shneider, Benjamin L; Sherker, Averell H; Sokol, Ronald J

    2015-01-01

    Objective Alpha-1-antitrypsin deficiency (A1AT) is a common genetic disease with unpredictable and highly variable course. The Childhood Liver Disease Research and Education Network (ChiLDREN) is an NIH, multi-center, longitudinal consortium studying pediatric liver diseases, with the objective of prospectively defining natural history and identifying disease modifiers. Methods Longitudinal, cohort study of A1AT patients birth through 25 years diagnosed with liver disease, type PIZZ or PISZ. Medical history, physical exam, laboratory, imaging, and standardized survey tool data were collected during the provision of standard of care. Results In this report of the cohort at baseline, 269 subjects were enrolled between Nov. 2008 and Oct. 2012 (208 with their native livers and 61 post-liver transplant). Subjects with mild disease (native livers and no portal hypertension [PHT]) compared to severe disease (with PHT or post-liver transplant) were not different in age at presentation. 57% of subjects with mild disease and 76% with severe disease were jaundiced at presentation (p=0.0024). 29% of subjects with native livers had PHT, but age at diagnosis and growth were not different between the no PHT and PHT groups (p>0.05). Subjects with native livers and PHT were more likely to have elevated bilirubin, ALT, AST, INR, and GGTP than the no PHT group (p≪0.001), but overlap was large. Chemistries alone could not identify PHT. Conclusion Many A1AT subjects presenting with elevated liver tests and jaundice improve spontaneously. Subjects with PHT have few symptoms and normal growth. Longitudinal cohort follow up will identify genetic and environmental disease modifiers. NCT00571272. PMID:25651489

  4. The Alpha-1 Association Genetic Counseling Program: an innovative approach to service.

    PubMed

    McGee, Dawn; Strange, Charlie; McClure, Rebecca; Schwarz, Laura; Erven, Marlene

    2011-08-01

    In an era of specialty medicine, genetic counselors are becoming increasingly focused in their service provision. The Alpha-1 Association Genetic Counseling Program, established in September 2007, specializes in confidential toll-free genetic counseling provided by a certified genetic counselor for Alpha-1 Antitrypsin deficiency, a co-dominant condition associated with lung and/or liver disease. The program received more than 600 callers in its first 2 years. Sixty-seven percent of new callers were family members, carriers, or health professionals. The number of callers increased between the first 2 years, with the greatest increases being family members and health professionals. Testing options and explanation of results encompassed 60% of initial reasons for calls. Seventy-two percent of referrals came from family and friends, test result letters, and the Alpha-1 Association. Between year 1 and 2 family member referrals showed the largest increase. This disease-specific genetic counseling program provides a model that may be useful for other rare disease communities.

  5. Alpha-1 couples: interpersonal and intrapersonal predictors of spousal communication and stress.

    PubMed

    Smith, Rachel A; Wienke, Sara; Coffman, Donna L

    2014-04-01

    Couples often discuss genetic test results, and then manage their implications together. This interdependence can lead to common, shared experiences, similar intrapersonal processes to manage shared stressors, or interpersonal influences between spouses, leading to different outcomes. This study sought to reveal the intracouple, intrapersonal, and interpersonal influences of genetic stigma and negative feelings on spousal communication and perceived stress with 50 couples in which one spouse is a member of a genetic disease registry. The results were analyzed with dyadic analysis, including multilevel modeling. The findings showed that registered members and their spouses were not statistically different in their mean levels of perceived genetic stigma, negative feelings about alpha-1 antitrypsin deficiency (AATD), conversations with each other about the AATD test results, and their perceived stress. The findings also showed that their intracouple consistencies were not high, and their intrapersonal and interpersonal influences on communication and stress differed. The social implications of genetic research at the interpersonal level are discussed. PMID:23934327

  6. Recent advances in the discovery of alpha1-adrenoceptor agonists.

    PubMed

    Bishop, Michael J

    2007-01-01

    The alpha(1) adrenoceptors are three of nine well-characterized receptors that are activated by epinephrine and norepinephrine. Agonists acting at the alpha(1) adrenoceptors produce numerous physiological effects, and are used therapeutically for several indications. Many known alpha(1) adrenoceptor agonists are alpha(1A) selective, but the discovery of highly selective alpha(1B) and alpha(1D) adrenoceptor agonists has proven to be an extremely difficult goal to achieve. This review will focus on recent advances in the discovery, development and clinical utility of subtype-specific alpha(1) agonists as well as contributions to our understanding of agonist-receptor interactions.

  7. Novel Treatment Strategies for Liver Disease Due to α1-Antitrypsin Deficiency

    PubMed Central

    Maurice, Nicholas; Perlmutter, David H.

    2014-01-01

    Alpha1-antitrypsin (AT) deficiency is the most common genetic cause of liver disease in children and is also a cause of chronic hepatic fibrosis, cirrhosis and hepatocellular carcinoma in adults. Recent advances in understanding how mutant AT molecules accumulate within hepatocytes and cause liver cell injury have led to a novel strategy for chemoprophylaxis of this liver disease. This strategy involves a class of drugs which enhance the intracellular degradation of mutant AT and, because several of these drugs have been used safely in humans for other indications, the strategy can be moved immediately into clinical trials. In this review we will also report on advances that provide a basis for several other strategies that could be used in the future for treatment of the liver disease associated with AT deficiency. PMID:22686209

  8. Protective effects of alpha1-acid glycoprotein and serum amyloid A on concanavalin A-induced liver failure via interleukin-6 induction by ME3738.

    PubMed

    Kuzuhara, Hiroyuki; Nakano, Yoshihisa; Yamashita, Nobuyuki; Imai, Masako; Kawamura, Yuji; Kurosawa, Tohru; Nishiyama, Shoji

    2006-07-17

    We examined whether the 22beta-methoxyolean-12-ene-3beta,24(4beta)-diol (ME3738)-mediated selective induction of interleukin-6 increased alpha1-acid glycoprotein and serum amyloid A expression, and whether these proteins protected against liver injury in vitro and in vivo. ME3738 treatment in male mice increased gene expression of alpha1-acid glycoprotein subtypes and serum amyloid A 2 genes, and plasma concentration of serum amyloid A. Treatment with alpha1-acid glycoprotein at 5 mg/animal or serum amyloid A at 0.03 and 0.1 mg/animal prior to concanavalin A administration reduced multifocal necrosis in the liver. Treatment with alpha1-acid glycoprotein and serum amyloid A, but not alpha1-antitrypsin, protected Hep G2 cells against cell injury. These results suggest that alpha1-acid glycoprotein and serum amyloid A, increased by ME3738-induced interleukin-6, might protect against concanavalin A-induced liver injury. PMID:16765939

  9. Alpha 1D- and alpha 1A-adrenoceptors mediate contraction in rat renal artery.

    PubMed

    Villalobos-Molina, R; López-Guerrero, J J; Ibarra, M

    1997-03-19

    To investigate the alpha 1-adrenoceptor subtype(s) mediating contraction in rat renal artery, we have compared the effect of the alpha 1-adrenoceptor antagonists, 5-methylurapidil, BMY 7378 (8-(2-(4-(2-methoxyphenyl)-1-piperazinyl) ethyl) 8-azaspiro (4.5) decane-7,9-dione 2HCl) and chloroethylclonidine on functional responses to noradrenaline. A clear blockade by chloroethylclonidine (10(-4) M) of noradrenaline-induced contraction was observed and, along with this effect. pKB values of 9.12 and 8.40 for BMY 7378 and 9.75 and 10.06 for 5-methylurapidil were obtained, indicating that the renal artery expresses the alpha 1D-adrenoceptor subtype as the one involved in contraction and not only the alpha 1A subtype as has been reported. PMID:9098691

  10. alpha(1A)- and alpha(1B)-adrenergic receptors differentially modulate antidepressant-like behavior in the mouse.

    PubMed

    Doze, Van A; Handel, Evelyn M; Jensen, Kelly A; Darsie, Belle; Luger, Elizabeth J; Haselton, James R; Talbot, Jeffery N; Rorabaugh, Boyd R

    2009-08-18

    Tricyclic antidepressant (TCA) drugs are used for the treatment of chronic depression, obsessive-compulsive disorder (OCD), and anxiety-related disorders. Chronic use of TCA drugs increases the expression of alpha(1)-adrenergic receptors (alpha(1)-ARs). Yet, it is unclear whether increased alpha(1)-AR expression contributes to the antidepressant effects of these drugs or if this effect is unrelated to their therapeutic benefit. In this study, mice expressing constitutively active mutant alpha(1A)-ARs (CAM alpha(1A)-AR) or CAM alpha(1B)-ARs were used to examine the effects of alpha(1A)- and alpha(1B)-AR signaling on rodent behavioral models of depression, OCD, and anxiety. CAM alpha(1A)-AR mice, but not CAM alpha(1B)-AR mice, exhibited antidepressant-like behavior in the tail suspension test and forced swim test. This behavior was reversed by prazosin, a selective alpha(1)-AR inverse agonist, and mimicked by chronically treating wild type mice with cirazoline, an alpha(1A)-AR agonist. Marble burying behavior, commonly used to model OCD in rodents, was significantly decreased in CAM alpha(1A)-AR mice but not in CAM alpha(1B)-AR mice. In contrast, no significant differences in anxiety-related behavior were observed between wild type, CAM alpha(1A)-AR, and CAM alpha(1B)-AR animals in the elevated plus maze and light/dark box. This is the first study to demonstrate that alpha(1A)- and alpha(1B)-ARs differentially modulate antidepressant-like behavior in the mouse. These data suggest that alpha(1A)-ARs may be a useful therapeutic target for the treatment of depression.

  11. Synthesis and evaluation of dioleoyl glyceric acids showing antitrypsin activity.

    PubMed

    Habe, Hiroshi; Fukuoka, Tokuma; Sato, Shun; Kitamoto, Dai; Sakaki, Keiji

    2011-01-01

    Previously, Lešová et al. reported the isolation and identification of metabolite OR-1, showing antitrypsin activity, produced during fermentation by Penicillium funiculosum. The structure of OR-1 was a mixture of glyceric acid (GA), esterified with C(14)-C(18) fatty acids, and oleic acid (C18:1) as the most predominant fatty acid (Folia Microbiol. 46, 21-23, 2001). In this study, dioleoyl D-GA and dioleoyl L-GA were synthesized via diesterification with oleoyl chloride, and their antitrypsin activities were evaluated using both a disk diffusion method and spectral absorption measurements. The results show that both compounds and their equivalent mixtures possess antitrypsin activities; however, their IC(50) values (approximately 2 mM) are much higher than that of OR-1 (4.25 µM), suggesting that dioleoyl GA does not play a major role in the OR-1 antitrypsin activity. PMID:21606621

  12. 8-NH2-boldine, an antagonist of alpha1A and alpha1B adrenoceptors without affinity for the alpha1D subtype: structural requirements for aporphines at alpha1-adrenoceptor subtypes.

    PubMed

    Ivorra, M Dolores; Valiente, Miguel; Martínez, Sonia; Madrero, Yolanda; Noguera, M Antonia; Cassels, Bruce K; Sobarzo, Eduardo M; D'Ocon, Pilar

    2005-10-01

    Structure-activity analysis of 21 aporphine derivatives was performed by examining their affinities for cloned human alpha (1A), alpha (1B) and alpha (1D) adrenoceptors (AR) using membranes prepared from rat-1 fibroblasts stably expressing each alpha (1)-AR subtype. All the compounds tested competed for [ (125)I]-HEAT binding with steep and monophasic curves. The most interesting compound was 8-NH (2)-boldine, which retains the selective affinity for alpha(1A)-AR (pKi = 6.37 +/- 0.21) vs. alpha(1B)-AR (pKi = 5.53 +/- 0.11) exhibited by 1,2,9,10-tetraoxygenated aporphines, but shows low affinity for alpha(1D)-AR (pKi < 2.5). Binding studies on native adrenoceptors present in rat cerebral cortex confirms the results obtained for human cloned alpha (1)-AR subtypes. The compounds selective for the alpha (1A) subtype discriminate two binding sites in rat cerebral cortex confirming a mixed population of alpha (1A)- and alpha (1B)-AR in this tissue. All compounds are more selective as inhibitors of [ (3)H]-prazosin binding than of [ (3)H]-diltiazem binding to rat cerebral cortical membranes. A close relationship was found between affinities obtained for cloned alpha (1A)-AR and inhibitory potencies on noradrenaline-induced contraction or inositol phosphate accumulation in tail artery, confirming that there is a homogeneous functional population of alpha(1A)-AR in this vessel. On the contrary, a poor correlation seems to exist between the affinity of 8-NH (2)-boldine for cloned alpha (1D)-AR and its potency as an inhibitor of noradrenaline-induced contraction or inositol phosphate accumulation in rat aorta, which confirms that a heterogeneous population of alpha (1)-AR mediates the adrenergic response in this vessel.

  13. Ozone inactivation of human alpha 1-proteinase inhibitor

    SciTech Connect

    Johnson, D.A.

    1980-06-01

    Ozone decreased the trypsin, chymotrypsin, and elastase inhibitory activities of human alpha 1-proteinase inhibitor both in plasma and in solutions of the pure inhibitor. The total loss of porcine elastase inhibitory activity required 18 mol of ozone/mol of pure alpha 1-PI and approximately 850 mol of ozone/mol of alpha 1-PI in plasma. A corresponding loss of the ability to inhibit human leukocyte elastase was observed. Inactivated alpha 1-PI contains four residues of methionine sulfoxide, in addition to oxidized tryosine and tryptophan. Electrophoretic analysis demonstrated that the ozone-inactivated alpha 1-PI did not form normal complexes with serine proteinases. These findings suggest that the inhalation of ozone could inactivate alpha 1-PI on the airspace side of the lung to create a localized alpha 1-PI deficiency, which might contribute to the development of emphysema.

  14. The Alpha-1A Adrenergic Receptor in the Rabbit Heart.

    PubMed

    Thomas, R Croft; Cowley, Patrick M; Singh, Abhishek; Myagmar, Bat-Erdene; Swigart, Philip M; Baker, Anthony J; Simpson, Paul C

    2016-01-01

    The alpha-1A-adrenergic receptor (AR) subtype is associated with cardioprotective signaling in the mouse and human heart. The rabbit is useful for cardiac disease modeling, but data on the alpha-1A in the rabbit heart are limited. Our objective was to test for expression and function of the alpha-1A in rabbit heart. By quantitative real-time reverse transcription PCR (qPCR) on mRNA from ventricular myocardium of adult male New Zealand White rabbits, the alpha-1B was 99% of total alpha-1-AR mRNA, with <1% alpha-1A and alpha-1D, whereas alpha-1A mRNA was over 50% of total in brain and liver. Saturation radioligand binding identified ~4 fmol total alpha-1-ARs per mg myocardial protein, with 17% alpha-1A by competition with the selective antagonist 5-methylurapidil. The alpha-1D was not detected by competition with BMY-7378, indicating that 83% of alpha-1-ARs were alpha-1B. In isolated left ventricle and right ventricle, the selective alpha-1A agonist A61603 stimulated a negative inotropic effect, versus a positive inotropic effect with the nonselective alpha-1-agonist phenylephrine and the beta-agonist isoproterenol. Blood pressure assay in conscious rabbits using an indwelling aortic telemeter showed that A61603 by bolus intravenous dosing increased mean arterial pressure by 20 mm Hg at 0.14 μg/kg, 10-fold lower than norepinephrine, and chronic A61603 infusion by iPRECIO programmable micro Infusion pump did not increase BP at 22 μg/kg/d. A myocardial slice model useful in human myocardium and an anthracycline cardiotoxicity model useful in mouse were both problematic in rabbit. We conclude that alpha-1A mRNA is very low in rabbit heart, but the receptor is present by binding and mediates a negative inotropic response. Expression and function of the alpha-1A in rabbit heart differ from mouse and human, but the vasopressor response is similar to mouse. PMID:27258143

  15. The Alpha-1A Adrenergic Receptor in the Rabbit Heart

    PubMed Central

    Myagmar, Bat-Erdene; Swigart, Philip M.; Baker, Anthony J.; Simpson, Paul C.

    2016-01-01

    The alpha-1A-adrenergic receptor (AR) subtype is associated with cardioprotective signaling in the mouse and human heart. The rabbit is useful for cardiac disease modeling, but data on the alpha-1A in the rabbit heart are limited. Our objective was to test for expression and function of the alpha-1A in rabbit heart. By quantitative real-time reverse transcription PCR (qPCR) on mRNA from ventricular myocardium of adult male New Zealand White rabbits, the alpha-1B was 99% of total alpha-1-AR mRNA, with <1% alpha-1A and alpha-1D, whereas alpha-1A mRNA was over 50% of total in brain and liver. Saturation radioligand binding identified ~4 fmol total alpha-1-ARs per mg myocardial protein, with 17% alpha-1A by competition with the selective antagonist 5-methylurapidil. The alpha-1D was not detected by competition with BMY-7378, indicating that 83% of alpha-1-ARs were alpha-1B. In isolated left ventricle and right ventricle, the selective alpha-1A agonist A61603 stimulated a negative inotropic effect, versus a positive inotropic effect with the nonselective alpha-1-agonist phenylephrine and the beta-agonist isoproterenol. Blood pressure assay in conscious rabbits using an indwelling aortic telemeter showed that A61603 by bolus intravenous dosing increased mean arterial pressure by 20 mm Hg at 0.14 μg/kg, 10-fold lower than norepinephrine, and chronic A61603 infusion by iPRECIO programmable micro Infusion pump did not increase BP at 22 μg/kg/d. A myocardial slice model useful in human myocardium and an anthracycline cardiotoxicity model useful in mouse were both problematic in rabbit. We conclude that alpha-1A mRNA is very low in rabbit heart, but the receptor is present by binding and mediates a negative inotropic response. Expression and function of the alpha-1A in rabbit heart differ from mouse and human, but the vasopressor response is similar to mouse. PMID:27258143

  16. Alpha 1-adrenoceptors mediating contraction in arteries of normotensive and spontaneously hypertensive rats are of the alpha 1D or alpha 1A subtypes.

    PubMed

    Villalobos-Molina, R; Ibarra, M

    1996-03-18

    Alpha 1-Adrenoceptor subtypes mediating contraction in carotid, aorta, mesenteric and caudal arteries from both Wistar Kyoto (WKY) normotensive and spontaneously hypertensive (SHR) rats were investigated by using the alpha 1A-adrenoceptor agonist methoxamine and antagonized with selective, competitive antagonists WB-4101, 5-methyl urapidil or BMY 7378 (8-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-8-azaspiro(4,5)decane -7,9-dione dihydrochloride). Isometric tension changes were recorded after methoxamine addition to the arterial rings, and the effects of the antagonists determined. All the antagonists shifted to the right the concentration-response curve to methoxamine. pA2 values indicate that all arteries but caudal express the alpha 1D-adrenoceptor subtype, since BMY 7378 values were high in these arteries. Due to the high pA2 values for 5-methyl urapidil and WB-4101 and the low values for BMY 7378 we conclude that the tail artery expresses the alpha 1A and not the alpha 1B subtype. No differences were found between both strains of rats, suggesting that hypertension does not modify the alpha 1-adrenoceptors in conductance arteries. PMID:8846824

  17. [Place of genotyping in addition to the phenotype and the assay of serum α-1 antitrypsin].

    PubMed

    Joly, Philippe; Francina, Alain; Lacan, Philippe; Heraut, Jessica; Chapuis-Cellier, Colette

    2011-01-01

    The diagnosis of deficiency of alpha-1 antitrypsin (A1AT) is based on isoelectric focusing of serum proteins and the extent of serum. However, the focusing is technically difficult and a greatly reduced concentration in abnormal A1AT tapeless does not differentiate an unstable variant of a variant called 'null' (that is to say without any phenotypic expression) to 'heterozygous' state. In this study, we compared the results of the assay, the phenotype and genotype of A1AT in 50 patients. Normal A1AT alleles (Pi*M1 to Pi*M4) or loss of the most common (Pi*S and Pi*Z) were clearly identified in phenotyping. However, genotyping was necessary to characterize: (i) certain alleles rarer A1AT (S-Munich, X-Christchurch); (ii) a null allele and; (iii) two new alleles A1AT not yet described in the literature. In conclusion, although the A1AT genotyping is generally not necessary, it is necessary to resolve complex cases and to obtain witnesses validated for isoelectric focusing.

  18. Adsorption of serum alpha-1-microglobulin onto biomaterials.

    PubMed

    Santin, M; Cannas, M; Wassall, M A; Denyer, S P

    1998-03-01

    The adsorption of alpha-1-microglobulin (alpha-1-m) from serum to the surface of polymers with different physicochemical properties was investigated. Enzyme-linked immunosorbent assay showed binding of this protein to the surface of polystyrene (PS), polyvinyl chloride (PVC) and a polyurethane, Chronoflex, after water washing, but only trace levels could be detected on two polymethacrylate derivatives, polymethyl methacrylate and poly(2-hydroxyethyl methacrylate). alpha-1-m was selectively desorbed from the five materials by sequential washes of serum-conditioned surfaces with isopropanol solutions at increasing concentrations. The presence of alpha-1-m in the washing supernatants was detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The relative binding strength of alpha-1-m to each surface was evaluated as the isopropanol (IsoPOH) concentration required to desorb the protein from that surface. Analysis of bound proteins by SDS-PAGE conclusively demonstrated the binding of a range of serum proteins, including alpha-1-m, to all polymer systems, but with varying binding strengths. The majority of protein was removed by water washing for the polymethacrylate polymers, while varying concentrations of IsoPOH were required to desorb proteins from PS, PVC and Chronoflex. There was a correlation between the hydrophobic nature of the material, determined by water contact angle measurements, and adsorption of alpha-1-m. Immunoblotting of isopropanol-eluted proteins by alpha-1-m antibodies showed the positive staining of a 29 kDa protein as well as selected bands within a molecular weight range of 40 200 kDa, suggesting the adsorption of this protein as both free and complexed forms. The ability of alpha-1-m to adsorb on to material surfaces and to participate in events relevant to the biocompatibility of a polymer, such as bacterial infection or inflammation control, suggests the need for further characterization of the properties of this

  19. [Medical expulsive therapy facilitated by alpha 1 adrenoceptor antagonist].

    PubMed

    Itoh, Yasunori; Niimi, Kazuhiro; Hirose, Yasuhiko

    2011-10-01

    In 2002, speedy elimination of ureterolithiasis in the lower part of ureter was first reported with the alpha 1 blocker. Thereafter, there are a lot of reports including meta-analysis about tamsulosin. In 2011 EAU Guidelines on Urolithiasis, it is the most important to establish effective MET (medical expulsive therapy) to facilitate spontaneous stone passage. Alpha 1 blockers are the preferred agents for MET. As a basic evidence for MET, we reported that alpha 1a and 1d AR subtype mRNA was highly expressed in the human ureter and that alpha 1A AR is the main participant in the human ureteral contraction. It is published newly in Japanese Guidelines on Urolithiasis revised edition to schedule to be published soon. PMID:21960238

  20. Elastase and alpha 1-protease inhibitor in burn wound exudates.

    PubMed

    Prager, M D; Herring, M; Germany, B; Baxter, C R

    1991-01-01

    By degrading antithrombin III, polymorphonuclear neutrophil (PMN) elastase can become a procoagulant. Because intravascular coagulation may accompany severe burn injury, this study examined burn wound exudates for PMN elastase and its physiologic inhibitor, plasma alpha 1-protease inhibitor (alpha 1-PI), as a step in evaluating their contributions to coagulopathy in patients with burns. Each of the nine exudates examined were inhibitory for PMN elastase. Chromatographic characterization of the inhibitor indicated that it was alpha 1-PI; its elution volume for four exudates was identical to that of pure alpha 1-PI. Confirmation of the inhibitor's identity was achieved by reaction of anti-alpha 1-PI antibody with each exudate and with inhibitory chromatographic fractions of exudates with the most inhibitory activity. Inhibitor potency, determined from dose-response curves against a standard PMN elastase activity, varied twentyfold among exudates. Only one exudate had catalytic activity with the PMN elastase substrate. Although this enzyme had elastase-like properties, it appeared to differ from PMN elastase. The presence of alpha 1-PI in the wound exudate suggests that this inhibitor may act to diminish fibrin formation from the level that might otherwise have been seen if excess elastase were free to degrade antithrombin III.

  1. alpha(1)-Microglobulin: a yellow-brown lipocalin.

    PubMed

    Akerström, B; Lögdberg, L; Berggård, T; Osmark, P; Lindqvist, A

    2000-10-18

    alpha(1)-Microglobulin, also called protein HC, is a lipocalin with immunosuppressive properties. The protein has been found in a number of vertebrate species including frogs and fish. This review summarizes the present knowledge of its structure, biosynthesis, tissue distribution and immunoregulatory properties. alpha(1)-Microglobulin has a yellow-brown color and is size and charge heterogeneous. This is caused by an array of small chromophore prosthetic groups, attached to amino acid residues at the entrance of the lipocalin pocket. A gene in the lipocalin cluster encodes alpha(1)-microglobulin together with a Kunitz-type proteinase inhibitor, bikunin. The gene is translated into the alpha(1)-microglobulin-bikunin precursor, which is subsequently cleaved and the two proteins secreted to the blood separately. alpha(1)-Microglobulin is found in blood and in connective tissue in most organs. It is most abundant at interfaces between the cells of the body and the environment, such as in lungs, intestine, kidneys and placenta. alpha(1)-Microglobulin inhibits immunological functions of white blood cells in vitro, and its distribution is consistent with an anti-inflammatory and protective role in vivo.

  2. alpha(1)-Adrenoceptors augment thermal hyperalgesia in mildly burnt skin.

    PubMed

    Drummond, Peter D

    2009-03-01

    The effect of the alpha(1)-adrenoceptor agonist phenylephrine on sensitivity to heat was investigated at three sites of mild burn injury in the cutaneous forearm of 19 healthy participants. Two of the sites were pre-treated with the alpha(1)-antagonist terazosin, to determine whether the effect of phenylephrine was mediated by alpha(1)-adrenoceptors. Terazosin was administered before the burn injury at one site, and after the burn injury at the other site. In another 15 participants, the nociceptive effect of the alpha(2)-adrenoceptor agonist clonidine was investigated with and without prior treatment with the alpha(2)-antagonist rauwolscine. Drugs were introduced into the skin by iontophoresis, and burns were induced by heating the skin to 48 degrees C for 2min. Heat pain thresholds to a temperature ramp (0.5 degrees C/s), and heat pain ratings to a thermal stimulus (45 degrees C, 7s), were determined before and after the administration of each drug. Thermal hyperalgesia provoked by phenylephrine was inhibited by terazosin administered after the burn injury, but not by terazosin administered before the burn injury. However, neither alpha(2)-adrenoceptor stimulation nor blockade affected sensitivity to heat in the mildly burnt skin. These findings suggest that stimulation of cutaneous alpha(1)-adrenoceptors increased the excitability of heat-sensitized nociceptive afferents. As terazosin was more effective when administered in burnt skin, an inflammatory response induced by the burn injury may have facilitated access of adrenergic agents to alpha(1)-adrenoceptors.

  3. The effects of SB 216469, an antagonist which discriminates between the alpha 1A-adrenoceptor and the human prostatic alpha 1-adrenoceptor.

    PubMed Central

    Chess-Williams, R.; Chapple, C. R.; Verfurth, F.; Noble, A. J.; Couldwell, C. J.; Michel, M. C.

    1996-01-01

    1. The affinity of the alpha 1-adrenoceptor antagonist SB 216469 (also known as REC 15/2739) has been determined at native and cloned alpha 1-adrenoceptor subtypes by radioligand binding and at functional alpha 1-adrenoceptor subtypes in isolated tissues. 2. In radioligand binding studies with [3H]-prazosin, SB 216469 had a high affinity at the alpha 1A-adrenoceptors of the rat cerebral cortex and kidney (9.5-9.8) but a lower affinity at the alpha 1B-adrenoceptors of the rat spleen and liver (7.7-8.2). 3. At cloned rat alpha 1-adrenoceptor subtypes transiently expressed in COS-1 cells and also at cloned human alpha 1-adrenoceptor subtypes stably transfected in Rat-1 cells, SB 216469 exhibited a high affinity at the alpha 1a-adrenoceptors (9.6-10.4) with a significantly lower affinity at the alpha 1b-adrenoceptor (8.0-8.4) and an intermediate affinity at the alpha 1d-adrenoceptor (8.7-9.2). 4. At functional alpha 1-adrenoceptors, SB 216469 had a similar pharmacological profile, with a high affinity at the alpha 1A-adrenoceptors of the rat vas deferens and anococcygeus muscle (pA2 = 9.5-10.0), a low affinity at the alpha 1B-adrenoceptors of the rat spleen (6.7) and guinea-pig aorta (8.0), and an intermediate affinity at the alpha 1D-adrenoceptors of the rat aorta (8.8). 5. Several recent studies have concluded that the alpha 1-adrenoceptor present in the human prostate has the pharmacological characteristics of the alpha 1A-adrenoceptor subtype. However, the affinity of SB 216469 at human prostatic alpha 1-adrenoceptors (pA2 = 8.1) determined in isolated tissue strips, was significantly lower than the values obtained at either the cloned alpha 1a-adrenoceptors (human, rat, bovine) or the native alpha 1A-adrenoceptors in radioligand binding and functional studies in the rat. 6. Our results with SB 216469, therefore, suggest that the alpha 1-adrenoceptor mediating contractile responses of the human prostate has properties which distinguish it from the cloned alpha 1a

  4. The alpha1-fetoprotein locus is activated by a nuclear receptor of the Drosophila FTZ-F1 family.

    PubMed

    Galarneau, L; Paré, J F; Allard, D; Hamel, D; Levesque, L; Tugwood, J D; Green, S; Bélanger, L

    1996-07-01

    The alpha1-fetoprotein (AFP) gene is located between the albumin and alpha-albumin genes and is activated by transcription factor FTF (fetoprotein transcription factor), presumed to transduce early developmental signals to the albumin gene cluster. We have identified FTF as an orphan nuclear receptor of the Drosophila FTZ-F1 family. FTF recognizes the DNA sequence 5'-TCAAGGTCA-3', the canonical recognition motif for FTZ-F1 receptors. cDNA sequence homologies indicate that rat FTF is the ortholog of mouse LRH-1 and Xenopus xFF1rA. Rodent FTF is encoded by a single-copy gene, related to the gene encoding steroidogenic factor 1 (SF-1). The 5.2-kb FTF transcript is translated from several in-frame initiator codons into FTF isoforms (54 to 64 kDa) which appear to bind DNA as monomers, with no need for a specific ligand, similar KdS (approximately equal 3 x 10(-10) M), and similar transcriptional effects. FTF activates the AFP promoter without the use of an amino-terminal activation domain; carboxy-terminus-truncated FTF exerts strong dominant negative effects. In the AFP promoter, FTF recruits an accessory trans-activator which imparts glucocorticoid reactivity upon the AFP gene. FTF binding sites are found in the promoters of other liver-expressed genes, some encoding liver transcription factors; FTF, liver alpha1-antitrypsin promoter factor LFB2, and HNF-3beta promoter factor UF2-H3beta are probably the same factor. FTF is also abundantly expressed in the pancreas and may exert differentiation functions in endodermal sublineages, similar to SF-1 in steroidogenic tissues. HepG2 hepatoma cells seem to express a mutated form of FTF.

  5. Automated docking of {alpha}-(1,4)- and {alpha}-(1,6)-linked glucosyl trisaccharides in the glucoamylase active site

    SciTech Connect

    Countinho, P.M.; Reilly, P.J.; Dowd, M.K.

    1998-06-01

    Low-energy conformers of five {alpha}-(1,4)- and {alpha}-(1,6)-linked glucosyl trisaccharides were flexibly docked into the glucoamylase active site using AutoDock 2.2. To ensure that all significant conformational space was searched, the starting trisaccharide conformers for docking were all possible combinations of the corresponding disaccharide low-energy conformers. All docked trisaccharides occupied subsites {minus}1 and +1 in very similar modes to those of corresponding nonreducing-end disaccharides. For linear substrates, full binding at subsite +2 occurred only when the substrate reducing end was {alpha}-(1,4)-linked, with hydrogen-bonding with the hydroxy-methyl group being the only polar interaction there. Given the absence of other important interactions at this subsite, multiple substrate conformations are allowed. For the one docked branched substrate, steric hindrance in the {alpha}-(1,6)-glycosidic oxygen suggests that the active-site residues have to change position for hydrolysis to occur. Subsite +1 of the glucoamylase active site allows flexibility in binding but, at least in Aspergillus glucoamylases, subsite +2 selectively binds substrates {alpha}-(1,4)-linked between subsites +1 and +2. Enzyme engineering to limit substrate flexibility at subsite +2 could improve glucoamylase industrial properties.

  6. 21 CFR 866.5080 - Alpha-1-antichymotrypsin immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... immunochemical techniques alpha-1-antichymotrypsin (a protein) in serum, other body fluids, and tissues. Alpha-1-antichymotrypsin helps protect tissues against proteolytic (protein-splitting) enzymes released during...

  7. Enlarged prostate - after care

    MedlinePlus

    BPH - self-care; Benign prostatic hypertrophy - self-care; Benign prostatic hyperplasia - self-care ... Your health care provider may have you take a medicine called alpha-1- blocker. Most people find that these drugs help ...

  8. [Application of alpha1-adrenoblockers in treatment of ureteral calculi].

    PubMed

    Dutov, V V; Popov, D V; Rumiantsev, A A; Pashchenko, V B

    2012-01-01

    The results of evaluation of the efficacy of alpha1-adrenoblockers in treatment of ureteral calculi are presented. Comparative, prospective, placebo-uncontrolled nonrandomized single-center study was performed, which included 118 patients with solitary diagnosed calculi in various parts of ureter. After the pain relief, all the patients underwent conservative therapy aimed at spontaneous discharge of concretions. The maximum duration of conservative treatment was 28 days. Ultrasound monitoring was performed every week in all patients. The control group of patients received only Drotaverinum 40 mg three times a day, and analgesics. The main group received alpha1-adrenoblocker tamsulosin at a standard dose of 0.4 mg once a day along with Drotaverinum and analgesics. The overall probability of a discharge of concrements localized in the distal ureter was significantly (P = 0.02) higher in the patients treated with alpha1-adrenoblockers. Treatment regimen in main group of patients allowed better control of pain during all periods of observation, even if the discharge of concretions was not registered. The overall probability of migration of concrements from the proximal to the distal ureter in main group of patients was 52% versus 32% in controls (P = 0.17). The frequency of adverse effects was comparable in both groups. Vertigo, postural hypotension, and weakness were significantly more frequent in the main group of patients. Univariate and multivariate analyses of the proportional hazards model have demonstrated that the administration of alpha1-adrenoblocker increased the likelihood of a discharge of concrement from the distal ureter. It is shown that the nature of the applied therapy has directly influence on the risk of an earlier discharge of concretions. Inclusion of alpha1-adrenoblockers in the treatment scheme increased the probability of discharge of concrements at 4.11 times.

  9. Preventing serpin aggregation: The molecular mechanism of citrate action upon antitrypsin unfolding

    SciTech Connect

    Pearce, Mary C.; Morton, Craig J.; Feil, Susanne C.; Hansen, Guido; Adams, Julian J.; Parker, Michael W.; Bottomley, Stephen P.

    2008-11-21

    The aggregation of antitrypsin into polymers is one of the causes of neonatal hepatitis, cirrhosis, and emphysema. A similar reaction resulting in disease can occur in other human serpins, and collectively they are known as the serpinopathies. One possible therapeutic strategy involves inhibiting the conformational changes involved in antitrypsin aggregation. The citrate ion has previously been shown to prevent antitrypsin aggregation and maintain the protein in an active conformation; its mechanism of action, however, is unknown. Here we demonstrate that the citrate ion prevents the initial misfolding of the native state to a polymerogenic intermediate in a concentration-dependent manner. Furthermore, we have solved the crystal structure of citrate bound to antitrypsin and show that a single citrate molecule binds in a pocket between the A and B beta-sheets, a region known to be important in maintaining antitrypsin stability.

  10. Ruptured Gastric Aneurysm in α-1 Antitrypsin Deficiency.

    PubMed

    Jaruvongvanich, Veeravich; Spanuchart, Ittikorn; Scott Gallacher, T

    2016-07-01

    We present a unique vascular complication of α-1 antitrypsin deficiency (AATD) in a patient with an acute onset of epigastric pain and hemodynamic instability. Abdominal computed tomography angiography detected hemoperitoneum and hematoma within the gastrohepatic ligament with active extravasation. Abdominal angiography revealed left gastric aneurysms. An association between AATD and vascular aneurysms has been suggested to be secondary to unopposed proteolytic activity against arterial structural proteins. The aneurysm formation in aortic, superior mesenteric, inferior mesenteric, and splenic arteries has been reported. We report the first case with ruptured gastric artery aneurysm as a complication of AATD. PMID:27622197

  11. Ruptured Gastric Aneurysm in α-1 Antitrypsin Deficiency

    PubMed Central

    Spanuchart, Ittikorn; Scott Gallacher, T.

    2016-01-01

    We present a unique vascular complication of α-1 antitrypsin deficiency (AATD) in a patient with an acute onset of epigastric pain and hemodynamic instability. Abdominal computed tomography angiography detected hemoperitoneum and hematoma within the gastrohepatic ligament with active extravasation. Abdominal angiography revealed left gastric aneurysms. An association between AATD and vascular aneurysms has been suggested to be secondary to unopposed proteolytic activity against arterial structural proteins. The aneurysm formation in aortic, superior mesenteric, inferior mesenteric, and splenic arteries has been reported. We report the first case with ruptured gastric artery aneurysm as a complication of AATD. PMID:27622197

  12. Oncodevelopmental and hormonal regulation of alpha 1-fetoprotein gene expression.

    PubMed

    Belanger, L; Baril, P; Guertin, M; Gingras, M C; Gourdeau, H; Anderson, A; Hamel, D; Boucher, J M

    1983-01-01

    The main features of the oncodevelopmental biology of alpha 1-fetoprotein (AFP) are reviewed. Progress made in the molecular biology of AFP gene regulation is discussed and we present our recent data on the mechanisms of AFP suppression by glucocorticoid hormones. The relationship between AFP gene transcription and cell replication is examined, and it is suggested that the degree of methylation of the AFP gene (or of co-methylated regulatory DNA sequences) conditions its response to hormones.

  13. Mechanisms of alpha 1-adrenergic vascular desensitization in conscious dogs

    NASA Technical Reports Server (NTRS)

    Kiuchi, K.; Vatner, D. E.; Uemura, N.; Bigaud, M.; Hasebe, N.; Hempel, D. M.; Graham, R. M.; Vatner, S. F.

    1992-01-01

    To investigate the mechanisms of alpha 1-adrenergic vascular desensitization, osmotic minipumps containing either saline (n = 9) or amidephrine mesylate (AMD) (n = 9), a selective alpha 1-adrenergic receptor agonist, were implanted subcutaneously in dogs with chronically implanted arterial and right atrial pressure catheters and aortic flow probes. After chronic alpha 1-adrenergic receptor stimulation, significant physiological desensitization to acute AMD challenges was observed, i.e., pressor and vasoconstrictor responses to the alpha 1-adrenergic agonist were significantly depressed (p < 0.01) compared with responses in the same dogs studied in the conscious state before pump implantation. However, physiological desensitization to acute challenges of the neurotransmitter norepinephrine (NE) (0.1 micrograms/kg per minute) in the presence of beta-adrenergic receptor blockade was not observed for either mean arterial pressure (MAP) (30 +/- 7 versus 28 +/- 5 mm Hg) or total peripheral resistance (TPR) (29.8 +/- 4.9 versus 28.9 +/- 7.3 mm Hg/l per minute). In the presence of beta-adrenergic receptor plus ganglionic blockade after AMD pump implantation, physiological desensitization to NE was unmasked since the control responses to NE (0.1 micrograms/kg per minute) before the AMD pumps were now greater (p < 0.01) than after chronic AMD administration for both MAP (66 +/- 5 versus 32 +/- 2 mm Hg) and TPR (42.6 +/- 10.3 versus 23.9 +/- 4.4 mm Hg/l per minute). In the presence of beta-adrenergic receptor, ganglionic, plus NE-uptake blockade after AMD pump implantation, desensitization was even more apparent, since NE (0.1 micrograms/kg per minute) induced even greater differences in MAP (33 +/- 5 versus 109 +/- 6 mm Hg) and TPR (28.1 +/- 1.8 versus 111.8 +/- 14.7 mm Hg/l per minute). The maximal force of contraction induced by NE in the presence or absence of endothelium was significantly decreased (p < 0.05) in vitro in mesenteric artery rings from AMD pump dogs

  14. Characterising the association of latency with α(1)-antitrypsin polymerisation using a novel monoclonal antibody.

    PubMed

    Tan, Lu; Perez, Juan; Mela, Marianna; Miranda, Elena; Burling, Keith A; Rouhani, Farshid N; DeMeo, Dawn L; Haq, Imran; Irving, James A; Ordóñez, Adriana; Dickens, Jennifer A; Brantly, Mark; Marciniak, Stefan J; Alexander, Graeme J M; Gooptu, Bibek; Lomas, David A

    2015-01-01

    α1-Antitrypsin is primarily synthesised in the liver, circulates to the lung and protects pulmonary tissues from proteolytic damage. The Z mutant (Glu342Lys) undergoes inactivating conformational change and polymerises. Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema. Latency is an analogous process of inactivating, intra-molecular conformational change and may co-occur with polymerisation. However, the relationship between latency and polymerisation remained unexplored in the absence of a suitable probe. We have developed a novel monoclonal antibody specific for latent α1-antitrypsin and used it in combination with a polymer-specific antibody, to assess the association of both conformers in vitro, in disease and during augmentation therapy. In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α1-antitrypsin. Polymers were extensively produced in hepatocytes and a cell line expressing Z α1-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway. However, α1-antitrypsin augmentation therapy contains latent α1-antitrypsin, as did the plasma of 63/274 PiZZ individuals treated with augmentation therapy but 0/264 who were not receiving this medication (p<10(-14)). We conclude that latent α1-antitrypsin is a by-product of the polymerisation pathway, that the intracellular folding environment is resistant to formation of the latent conformer but that augmentation therapy introduces latent α1-antitrypsin into the circulation. A suite of monoclonal antibodies and methodologies developed in this study can characterise α1-antitrypsin folding and conformational transitions, and screen methods to improve augmentation therapy. PMID:25462157

  15. Characterising the association of latency with α1-antitrypsin polymerisation using a novel monoclonal antibody

    PubMed Central

    Tan, Lu; Perez, Juan; Mela, Marianna; Miranda, Elena; Burling, Keith A; Rouhani, Farshid N; DeMeo, Dawn L; Haq, Imran; Irving, James A; Ordóñez, Adriana; Dickens, Jennifer A; Brantly, Mark; Marciniak, Stefan J; Alexander, Graeme J M; Gooptu, Bibek; Lomas, David A

    2015-01-01

    α1-Antitrypsin is primarily synthesised in the liver, circulates to the lung and protects pulmonary tissues from proteolytic damage. The Z mutant (Glu342Lys) undergoes inactivating conformational change and polymerises. Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema. Latency is an analogous process of inactivating, intra-molecular conformational change and may co-occur with polymerisation. However, the relationship between latency and polymerisation remained unexplored in the absence of a suitable probe. We have developed a novel monoclonal antibody specific for latent α1-antitrypsin and used it in combination with a polymer-specific antibody, to assess the association of both conformers in vitro, in disease and during augmentation therapy. In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α1-antitrypsin. Polymers were extensively produced in hepatocytes and a cell line expressing Z α1-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway. However, α1-antitrypsin augmentation therapy contains latent α1-antitrypsin, as did the plasma of 63/274 PiZZ individuals treated with augmentation therapy but 0/264 who were not receiving this medication (p < 10−14). We conclude that latent α1-antitrypsin is a by-product of the polymerisation pathway, that the intracellular folding environment is resistant to formation of the latent conformer but that augmentation therapy introduces latent α1-antitrypsin into the circulation. A suite of monoclonal antibodies and methodologies developed in this study can characterise α1-antitrypsin folding and conformational transitions, and screen methods to improve augmentation therapy. PMID:25462157

  16. Characterising the association of latency with α(1)-antitrypsin polymerisation using a novel monoclonal antibody.

    PubMed

    Tan, Lu; Perez, Juan; Mela, Marianna; Miranda, Elena; Burling, Keith A; Rouhani, Farshid N; DeMeo, Dawn L; Haq, Imran; Irving, James A; Ordóñez, Adriana; Dickens, Jennifer A; Brantly, Mark; Marciniak, Stefan J; Alexander, Graeme J M; Gooptu, Bibek; Lomas, David A

    2015-01-01

    α1-Antitrypsin is primarily synthesised in the liver, circulates to the lung and protects pulmonary tissues from proteolytic damage. The Z mutant (Glu342Lys) undergoes inactivating conformational change and polymerises. Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema. Latency is an analogous process of inactivating, intra-molecular conformational change and may co-occur with polymerisation. However, the relationship between latency and polymerisation remained unexplored in the absence of a suitable probe. We have developed a novel monoclonal antibody specific for latent α1-antitrypsin and used it in combination with a polymer-specific antibody, to assess the association of both conformers in vitro, in disease and during augmentation therapy. In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α1-antitrypsin. Polymers were extensively produced in hepatocytes and a cell line expressing Z α1-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway. However, α1-antitrypsin augmentation therapy contains latent α1-antitrypsin, as did the plasma of 63/274 PiZZ individuals treated with augmentation therapy but 0/264 who were not receiving this medication (p<10(-14)). We conclude that latent α1-antitrypsin is a by-product of the polymerisation pathway, that the intracellular folding environment is resistant to formation of the latent conformer but that augmentation therapy introduces latent α1-antitrypsin into the circulation. A suite of monoclonal antibodies and methodologies developed in this study can characterise α1-antitrypsin folding and conformational transitions, and screen methods to improve augmentation therapy.

  17. Alpha1-adrenoreceptor in human hippocampus: binding and receptor subtype mRNA expression.

    PubMed

    Szot, Patricia; White, Sylvia S; Greenup, J Lynne; Leverenz, James B; Peskind, Elaine R; Raskind, Murray A

    2005-10-01

    Alpha1-adrenoreceptors (AR), of which three subtypes exist (alpha1A-, alpha1B- and alpha1D-AR) are G-protein-coupled receptors that mediate the actions of norepinephrine and epinephrine both peripherally and centrally. In the CNS, alpha1-ARs are found in the hippocampus where animal studies have shown the ability of alpha1-AR agents to modulate long-term potentiation and memory; however, the precise distribution of alpha1-AR expression and its subtypes in the human brain is unknown making functional comparisons difficult. In the human hippocampus, 3H-prazosin (alpha1-AR antagonist) labels only the dentate gyrus (molecular, granule and polymorphic layers) and the stratum lucidum of the CA3 homogeneously. Human alpha1A-AR mRNA in the hippocampus is observed only in the dentate gyrus granule cell layer, while alpha1D-AR mRNA expression is observed only in the pyramidal cell layers of CA1, CA2 and CA3, regions where 3H-prazosin did not bind. alpha1B-AR mRNA is not expressed at detectable levels in the human hippocampus. These results confirm a difference in hippocampal alpha1-AR localization between rat and humans and further describe a difference in the localization of the alpha1A- and alpha1D-AR mRNA subtype between rats and humans. PMID:16039007

  18. Evaluation of the pharmacological selectivity profile of alpha 1 adrenoceptor antagonists at prostatic alpha 1 adrenoceptors: binding, functional and in vivo studies.

    PubMed Central

    Kenny, B. A.; Miller, A. M.; Williamson, I. J.; O'Connell, J.; Chalmers, D. H.; Naylor, A. M.

    1996-01-01

    1. The profile of a range of alpha 1 adrenoceptor antagonists was determined in vitro against cloned human alpha 1A, alpha 1B and alpha 1D adrenoceptors and against noradrenaline-mediated contractions of rat aorta and human prostate. The in vivo profile of compounds was determined in an anaesthetized dog model which allowed the simultaneous assessment of antagonist potency against phenylephrine-mediated increases in blood pressure and prostatic pressure. 2. The quinazoline antagonists, prazosin, doxazosin and alfuzosin displayed high affinity but were non selective for the three cloned human alpha 1 adrenoceptors. Indoramin and SNAP 1069 showed selectivity for alpha 1A and alpha 1B adrenoceptors relative to the alpha 1D subtype. Rec 15/2739, WB 4101, SL 89,0591, (+)- and (-)- tamsulosin showed selectivity for alpha 1A and alpha 1D adrenoceptors relative to the alpha 1B subtype. RS 17053 showed high affinity and selectivity for alpha 1A adrenoceptors (pKi 8.6) relative to alpha 1B (pKi = 7.3) and alpha 1D (pKi = 7.1) subtypes. 3. (+)-Tamsulosin, (-)-tamsulosin, SL 89,0591, Rec 15/2739, SNAP 1069 and RS 17053 appeared to act as competitive antagonists of noradrenaline-mediated contractions of rat aorta yielding pA2 affinity estimates which were similar to binding affinities at cloned human alpha 1D adrenoceptors. The following rank order was obtained: prazosin = (-)-tamsulosin > doxazosin > SL 89,0591 = (+)-tamsulosin > Rec 15/2739 > RS 17053 = SNAP 1069. 4. (-)-Tamsulosin was a very potent, insurmountable antagonist of noradrenaline-mediated contractions of human prostate, yielding an approximate pA2 estimate of 9.8 at 1 nM. The corresponding (+)-enantiomer was 30 fold weaker. SL 89,0591, SNAP 1069 and Rec 15/2739 yielded pA2 estimates which compared well with their alpha 1A binding affinities. The affinity estimate for prazosin on human prostate was lower than the corresponding binding affinity determined at alpha 1A adrenoceptors and RS 17053 was a very weak

  19. Alpha1-antichymotrypsin polymorphism in Japanese cases of Alzheimer's disease.

    PubMed

    Yoshizawa, T; Yamakawa-Kobayashi, K; Hamaguchi, H; Shoji, S

    1997-11-25

    We examined the possible involvement of alpha1-antichymotrypsin (ACT) polymorphism in the risk for Alzheimer's disease (AD) in a Japanese population. No differences between AD and control subjects have been shown in the genotype distributions and allele frequencies of ACT. No modification of the risk for AD was observed, either alone or in combination with the apolipoprotein epsilon4 (ApoE-4) allele. Our results from a Japanese population failed to confirm the previous data in which the ACT polymorphism was shown to affect the ApoE-4-associated risk for AD.

  20. 21 CFR 866.5580 - Alpha-1-lipoprotein immuno-logical test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... the alpha-1-lipoprotein (high-density lipoprotein) in serum and plasma. Measurement of alpha-1-lipoprotein may aid in the diagnosis of Tangier disease (a hereditary disorder of fat metabolism)....

  1. 21 CFR 866.5580 - Alpha-1-lipoprotein immuno-logical test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... the alpha-1-lipoprotein (high-density lipoprotein) in serum and plasma. Measurement of alpha-1-lipoprotein may aid in the diagnosis of Tangier disease (a hereditary disorder of fat metabolism)....

  2. Alfa-one antitrypsin phenotypes and inhalatory pulmonary pathology.

    PubMed

    Amaral-Marques, R; Avila, R; Geada, H; Cochito, M L; Manso, C; Villar, T G

    1980-01-01

    One hundred and twenty nine workers in the cork industry, 69 rural workers, 66 carpet makers, 58 workers in a granite quarry and 51 workers in a rice husking factory were studied from an epidemiologic point of view. All were submitted to a standard questionnaire planned to detect respiratory disease due to inhalatory causes. They were submitted to a clinical examination, summary ventilatory function tests, a 70 mm microradiograph, and blood was taken to determine alfa-one antitrypsin and its phenotypes and, in the cork industry workers and rice husking workers, the level of IgA, IgG and IgM. The results are presented and an attempt is made to correlate the various parameters among themselves, and namely alfa-one AT phenotypes with the existence of respiratory pathology. Finally the results are discussed.

  3. '1-Antitrypsin polymorphism and systematics of eastern North American wolves

    USGS Publications Warehouse

    Mech, L.D.; Federoff, N.E.

    2002-01-01

    We used data on the polymorphic status of '1-antitrypsin ('1AT) to study the relationship of Minnesota wolves to the gray wolf (Canis lupus), which was thought to have evolved in Eurasia, and to red wolves (Canis rufus) and coyotes (Canis latrans), which putatively evolved in North America. Recent evidence had indicated that Minnesota wolves might be more closely related to red wolves and coyotes. Samples from wild-caught Minnesota wolves and from captive wolves, at least some of which originated in Alaska and western Canada, were similarly polymorphic for '1AT, whereas coyote and red wolf samples were all monomorphic. Our findings, in conjunction with earlier results, are consistent with the Minnesota wolf being a gray wolf of Eurasian origin or possibly a hybrid between the gray wolf of Eurasian origin and the proposed North American wolf.

  4. Longer telomere length in COPD patients with α1-antitrypsin deficiency independent of lung function.

    PubMed

    Saferali, Aabida; Lee, Jee; Sin, Don D; Rouhani, Farshid N; Brantly, Mark L; Sandford, Andrew J

    2014-01-01

    Oxidative stress is involved in the pathogenesis of airway obstruction in α1-antitrypsin deficient patients. This may result in a shortening of telomere length, resulting in cellular senescence. To test whether telomere length differs in α1-antitrypsin deficient patients compared with controls, we measured telomere length in DNA from peripheral blood cells of 217 α1-antitrypsin deficient patients and 217 control COPD patients. We also tested for differences in telomere length between DNA from blood and DNA from lung tissue in a subset of 51 controls. We found that telomere length in the blood was significantly longer in α1-antitrypsin deficient COPD patients compared with control COPD patients (p = 1×10(-29)). Telomere length was not related to lung function in α1-antitrypsin deficient patients (p = 0.3122) or in COPD controls (p = 0.1430). Although mean telomere length was significantly shorter in the blood when compared with the lungs (p = 0.0078), telomere length was correlated between the two tissue types (p = 0.0122). Our results indicate that telomere length is better preserved in α1-antitrypsin deficient COPD patients than in non-deficient patients. In addition, measurement of telomere length in the blood may be a suitable surrogate for measurement in the lung.

  5. Modeling the interactions between alpha(1)-adrenergic receptors and their antagonists.

    PubMed

    Du, Lupei; Li, Minyong

    2010-09-01

    As crucial members of the G-protein coupled receptor (GPCR) superfamily, alpha (1)-adrenergic receptors (alpha(1)-ARs) are recognized to intervene the actions of endogenous catecholamines such as norepinephrine and epinephrine. So far three distinct alpha(1)-AR subtypes, alpha(1A), alpha(1B) and alpha(1D), have been characterized by functional analysis, radio-ligand binding and molecular biology studies. The alpha(1)-ARs are of therapeutic interest because of their distinct and critical roles in many physiological processes, containing hypertension, benign prostatic hyperplasia, smooth muscle contraction, myocardial inotropy and chronotropy, and hepatic glucose metabolism. Accordingly, designing subtype-selective antagonists for each of the three alpha(1)-AR subtypes has been an enthusiastic region of medicinal research. Even though a large number of studies on GPCRs have been conducted, understanding of how known antagonists bind to alpha(1)-ARs still remains sketchy and has been a serious impediment to search for potent and subtype-selective alpha(1)-AR antagonists because of the lack of detailed experimental structural knowledge. This review deliberates the simulation of alpha(1)-ARs and their interactions with antagonists by using ligand-based (pharmacophore identification and QSAR modeling) and structure-based (comparative modeling and molecular docking) approaches. Combined with experimental data, these computational attempts could improve our understanding of the structural basis of antagonist binding and the molecular basis of receptor activation, thus offering a more reasonable approach in the design of drugs targeting alpha(1)-ARs.

  6. 21 CFR 866.5580 - Alpha-1-lipoprotein immuno-logical test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5580 Alpha-1-lipoprotein immuno-logical test system. (a) Identification. An alpha-1-lipoprotein... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alpha-1-lipoprotein immuno-logical test...

  7. 21 CFR 866.5580 - Alpha-1-lipoprotein immuno-logical test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5580 Alpha-1-lipoprotein immuno-logical test system. (a) Identification. An alpha-1-lipoprotein... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alpha-1-lipoprotein immuno-logical test...

  8. Implication of alpha1-antichymotrypsin polymorphism in familial Alzheimer's disease.

    PubMed

    Nacmias, B; Marcon, G; Tedde, A; Forleo, P; Latorraca, S; Piacentini, S; Amaducci, L; Sorbi, S

    1998-03-13

    A common polymorphism in the alpha1-antichymotrypsin (ACT) gene has been shown to modify the Apolipoprotein E (ApoE) epsilon4-associated Alzheimer's disease (AD) risk identifying the combination of the ACT/AA and ApoE epsilon4/epsilon4 genotypes as a potential susceptibility marker for AD. Using the polymerase chain reaction, we analyzed the segregation of the ACT and ApoE polymorphisms in familial Alzheimer's disease (FAD) patients carrying mutations in Presenilin (PS) and APP genes and in both early onset (EO) and late onset (LO) FAD patients without known mutations. Our data suggest that ACT does not represent an additional risk factor for PS and APP mutated families. However, in LOFAD patients a high frequency of the combined ACT/AA and ApoE epsilon4/epsilon4 genotypes suggest that ACT may interact with ApoE and play a role in LOFAD. PMID:9572591

  9. Apolipoprotein E and alpha1-antichymotrypsin polymorphism in Alzheimer's disease.

    PubMed

    Nacmias, B; Tedde, A; Latorraca, S; Piacentini, S; Bracco, L; Amaducci, L; Guarnieri, B M; Petruzzi, C; Ortenzi, L; Sorbi, S

    1996-10-01

    A recent observation has shown that a common polymorphism in the alpha1-antichymotrypsin (ACT) gene modifies the apolipoprotein E (ApoE) epsilon4-associated Alzheimer's disease (AD) risk identifying the combination of the ACT/AA and ApoE epsilon4/epsilon4 genotypes as a potential susceptibility marker for AD. We analyzed the segregation of the ApoE and ACT polymorphism in sporadic and familial AD patients. In none of the sporadic AD patients did we find the combination of the ACT/AA and ApoE epsilon4/epsilon4 genotypes. The frequency of ApoE epsilon4/epsilon4 homozygosity in the AD sample resulted highest for the ACT/ TT genotype (17.6%). Our data fail to confirm any additional association with AD beyond the ApoE epsilon4 allele with any ACT genotype, suggesting that ACT does not represent an additional risk factor for AD. PMID:8871590

  10. The action of calcium channel blockers on recombinant L-type calcium channel alpha1-subunits.

    PubMed

    Morel, N; Buryi, V; Feron, O; Gomez, J P; Christen, M O; Godfraind, T

    1998-11-01

    1. CHO cells expressing the alpha(1C-a) subunit (cardiac isoform) and the alpha(1C-b) subunit (vascular isoform) of the voltage-dependent L-type Ca2+ channel were used to investigate whether tissue selectivity of Ca2+ channel blockers could be related to different affinities for alpha1C isoforms. 2. Inward current evoked by the transfected alpha1 subunit was recorded by the patch-clamp technique in the whole-cell configuration. 3. Neutral dihydropyridines (nifedipine, nisoldipine, (+)-PN200-110) were more potent inhibitors of alpha(1C-)b-subunit than of alpha(1C-a)-subunit. This difference was more marked at a holding potential of -100 mV than at -50 mV. SDZ 207-180 (an ionized dihydropyridine) exhibited the same potency on the two isoforms. 4. Pinaverium (ionized non-dihydropyridine derivative) was 2 and 4 fold more potent on alpha(1C-a) than on alpha(1C-b) subunit at Vh of -100 mV and -50 mV, respectively. Effects of verapamil were identical on the two isoforms at both voltages. 5. [3H]-(+)-PN 200-110 binding experiments showed that neutral dihydropyridines had a higher affinity for the alpha(1C-b) than for the alpha(1C-a) subunit. SDZ 207-180 had the same affinity for the two isoforms and pinaverium had a higher affinity for the alpha(1C-a) subunit than for the alpha(1C-b) subunit. 6. These results indicate marked differences among Ca2+ channel blockers in their selectivity for the alpha(1C-a) and alpha(1C-b) subunits of the Ca2+ channel. PMID:9846638

  11. Biosynthesis and secretion of alpha 1 acute-phase globulin in primary cultures of rat hepatocytes.

    PubMed

    Bauer, J; Kurdowska, A; Tran-Thi, T A; Budek, W; Koj, A; Decker, K; Heinrich, P C

    1985-01-15

    Experimental inflammation in rats led to a sevenfold increase in serum levels of alpha 1 acute-phase globulin. This increase is correlated with elevated levels of translatable mRNA for alpha 1 acute-phase globulin in the liver. Biosynthesis and secretion of alpha 1 acute-phase globulin were studied in rat hepatocyte primary cultures. An intracellular form of alpha 1 acute-phase globulin with an apparent relative molecular mass of 63 500 and a secreted form of 68 000 were found. The intracellular form of alpha 1 acute-phase globulin could be deglycosylated by endoglucosaminidase H treatment indicating that its oligosaccharide chains were of the high-mannose type. The secreted form of alpha 1 acute-phase globulin was not sensitive to endoglucosaminidase H, but was susceptible to the action of sialidase reflecting carbohydrate side-chains of the complex type. Pulse-chase experiments revealed a precursor-product relationship for the high-mannose and the complex type alpha 1 acute-phase globulin. In the hepatocyte medium newly synthesized alpha 1 acute-phase globulin was detected 30 min after the pulse. Unglycosylated alpha 1 acute-phase globulin was found in the cells as well as in the medium when the transfer of oligosaccharide chains onto the polypeptide chains was blocked by tunicamycin. Tunicamycin led to a marked delay in alpha 1 acute-phase globulin secretion. PMID:2578391

  12. Role of alpha-1 adrenoceptor subtypes mediating constriction of the rabbit ear thermoregulatory microvasculature.

    PubMed

    Li, Z; Silver, W P; Koman, L A; Strandhoy, J W; Rosencrance, E; Gordon, S; Smith, T L

    2000-01-01

    An acute in vivo preparation of the microvasculature of the rabbit ear was used to evaluate the functional role of alpha1 (alpha1)-adrenoceptor subtypes in thermoregulatory microcirculation. The effect of alpha1-adrenoceptor subtype blockade on phenylephrine-induced vasoconstriction was assessed with the alpha1A, alpha1B, and alpha1D-adrenoceptor-selective antagonists 5-methyl-urapidil (10(-8) M), chloroethylclonidine (10(-5) M), and 8-[2-[4(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspirol[4.5]deca ne-7,9-dione dihydrochloride (BMY7378) (10(-6) M), respectively. The results demonstrated that pretreatment of the ear microvasculature with 5-methyl-urapidil or BMY7378 shifted the phenylephrine concentration-response curve rightward and significantly changed the log of the phenylephrine concentration, causing half-maximum stimulation (EC50) in arterioles (p < 0.05). BMY7378 shifted the phenylephrine concentration-response curve of the arteriovenous anastomoses about 100-fold rightward (p < 0.05). All three alpha1-adrenoceptor antagonists eliminated the vasoconstrictive effects of phenylephrine on venules. The results indicate that the ear microvasculature has a heterogenous distribution of alpha1-adrenoceptor subtypes. The alpha1A and alpha1D-adrenoceptor subtypes appear to have a greater influence on constrictive function in arterioles, whereas the alpha1D-adrenoceptor is the dominant constrictor of arteriovenous anastomoses. In general, the alpha1-adrenoceptor does not play a major vasoconstrictor role in venules. Chloroethylclonidine, an irreversible alpha1B-adrenoceptor antagonist, induced contractile responses in the ear microvasculature, probably due to its alpha2-adrenoceptor agonist effects. This study extended our understanding of the adrenergic receptor control mechanisms of a cutaneous thermoregulatory end organ characterized by two parallel perfusion circuits providing nutritional and thermoregulatory functions. PMID:10716292

  13. Functional evidence equating the pharmacologically-defined alpha 1A- and cloned alpha 1C-adrenoceptor: studies in the isolated perfused kidney of rat.

    PubMed Central

    Blue, D. R.; Bonhaus, D. W.; Ford, A. P.; Pfister, J. R.; Sharif, N. A.; Shieh, I. A.; Vimont, R. L.; Williams, T. J.; Clarke, D. E.

    1995-01-01

    1. The present study characterizes and classifies alpha 1-adrenoceptor-mediated vasoconstriction in the isolated perfused kidney of rat using quantitative receptor pharmacology and compares the results to radioligand binding studies (made in cloned alpha 1-adrenoceptor subtypes, native alpha 1A-adrenoceptors in submaxillary gland of rat, and alpha 1A-adrenoceptors in several other tissues of rat). 2. Concentration-effect curves to noradrenaline in the presence of 5-methyl-urapidil were biphasic, indicating alpha 1-adrenoceptor heterogeneity. The alpha 1-adrenoceptor subtype mediating the first phase (low affinity for 5-methyl-urapidil) could not be 'isolated' for detailed pharmacological characterization but was defined by a sensitivity to inhibition by chloroethylclonidine and an inability of methoxamine to activate the site. Additionally, vasoconstriction mediated by this alpha 1-adrenoceptor subtype or subtypes was abolished by nitrendipine (1 microM), thereby allowing characterization of the second, high affinity site for 5-methyl-urapidil. 3. The following antagonists interacted competitively with noradrenaline at the alpha 1-adrenoceptor for which 5-methyl-urapidil exhibits high affinity (pKB value): WB 4101 (10.3) > prazosin (9.5) approximately HV 723 (9.3) approximately 5-methyl-urapidil (9.2) > phenotolamine (8.6) > spiperone (pA2 = 8.1) approximately oxymetazoline (7.9). In contrast, insurmountable antagonism was seen with S(+)- and R(-)-niguldipine, the S(+)-isomer being approximately 30 fold more potent than the R(-)-isomer. Receptor protection experiments indicated that S(+)-niguldipine interacted directly with alpha 1-adrenoceptors. Dehydroniguldipine acted as a competitive antagonist (pKB = 9.0). Thus, the results with antagonists define the alpha 1-adrenoceptor as an alpha 1A-adrenoceptor. 4. An agonist 'fingerprint' was constructed in the presence of nitrendipine to define further the alpha 1A-adrenoceptor. The following order and relativity of

  14. Increase of mouse resistance to Candida albicans infection by thymosin alpha 1.

    PubMed Central

    Bistoni, F; Marconi, P; Frati, L; Bonmassar, E; Garaci, E

    1982-01-01

    Studies were carried out to assess the ability of thymosin alpha 1 to prolong the survival of mice challenged with Candida albicans. Two- to four-month-old mice were treated with graded doses of thymosin alpha 1 before, after, or before and after intravenous challenge with C. albicans. Significant resistance ot lethal infection was afforded by 100 micrograms of thymosin alpha 1 per kg given before or before and after challenge, whereas no protection was found in mice treated with thymosin alpha 1 administered at any dose level after inoculation. Pretreatment with thymosin alpha 1 also prevented the increased susceptibility to C. albicans infection of mice pretreated with cyclophosphamide on day -6. The results showed that thymosin alpha 1 was capable of protecting untreated or cyclophosphamide-pretreated mice from C. albicans infection at an optimal dose and schedule of administration. PMID:7085074

  15. A lymphokine regulates expression of alpha-1-proteinase inhibitor in human monocytes and macrophages.

    PubMed Central

    Takemura, S; Rossing, T H; Perlmutter, D H

    1986-01-01

    Biosynthesis and secretion of alpha-1-proteinase inhibitor (alpha 1 PI) has been demonstrated in primary cultures of human mononuclear phagocytes, making it possible to study regulation of alpha 1 PI in normal (PiMM) and homozygous-deficient (PiZZ) individuals. In this study, expression of alpha 1 PI by blood monocytes, bronchoalveolar, and breast milk macrophages decreased during 1 wk in culture whereas expression of other secreted proteins increased. The addition of crude supernatants from mitogen-stimulated peripheral blood mononuclear cells to confluent monolayers of mononuclear phagocytes after 1 wk in culture resulted in a 2- to 2.5-fold increase in alpha 1 PI expression. The increase in alpha 1 PI expression was dose- and time-dependent, and involved a mechanism acting at a pretranslational level as shown by an increase in specific messenger RNA content corresponding to the increase in synthesis and secretion of alpha 1 PI. Although alpha 1 PI was expressed in native form and in forms complexed with serine protease by monocytes early in culture, it was expressed in its native form alone when monocytes were incubated with the lymphokine after 1 wk in culture. The regulating factor had the characteristics of a polypeptide and was derived from T lymphocytes, but it was not interferon-alpha, -beta, -gamma, or interleukin 2. This lymphokine also stimulated synthesis of alpha 1 PI in monocytes of homozygous-deficient PiZZ individuals, but had minimal effect on secretion, thereby increasing the intracellular accumulation of the inhibitor and exaggerating the defect in secretion of alpha 1 PI in these individuals. Regulation of mononuclear phagocyte alpha 1 PI expression by a lymphokine provides a model for further analysis of the effect of enhanced synthesis on a defect in posttranslational processing/secretion and for analysis of differential regulation of protease and inhibitor expressed in the same cells. Images PMID:3485658

  16. Molecular Mechanism of Z α1-Antitrypsin Deficiency.

    PubMed

    Huang, Xin; Zheng, Ying; Zhang, Fei; Wei, Zhenquan; Wang, Yugang; Carrell, Robin W; Read, Randy J; Chen, Guo-Qiang; Zhou, Aiwu

    2016-07-22

    The Z mutation (E342K) of α1-antitrypsin (α1-AT), carried by 4% of Northern Europeans, predisposes to early onset of emphysema due to decreased functional α1-AT in the lung and to liver cirrhosis due to accumulation of polymers in hepatocytes. However, it remains unclear why the Z mutation causes intracellular polymerization of nascent Z α1-AT and why 15% of the expressed Z α1-AT is secreted into circulation as functional, but polymerogenic, monomers. Here, we solve the crystal structure of the Z-monomer and have engineered replacements to assess the conformational role of residue Glu-342 in α1-AT. The results reveal that Z α1-AT has a labile strand 5 of the central β-sheet A (s5A) with a consequent equilibrium between a native inhibitory conformation, as in its crystal structure here, and an aberrant conformation with s5A only partially incorporated into the central β-sheet. This aberrant conformation, induced by the loss of interactions from the Glu-342 side chain, explains why Z α1-AT is prone to polymerization and readily binds to a 6-mer peptide, and it supports that annealing of s5A into the central β-sheet is a crucial step in the serpins' metastable conformational formation. The demonstration that the aberrant conformation can be rectified through stabilization of the labile s5A by binding of a small molecule opens a potential therapeutic approach for Z α1-AT deficiency. PMID:27246852

  17. Molecular Mechanism of Z α1-Antitrypsin Deficiency*

    PubMed Central

    Huang, Xin; Zheng, Ying; Zhang, Fei; Wei, Zhenquan; Wang, Yugang; Carrell, Robin W.; Read, Randy J.; Chen, Guo-Qiang; Zhou, Aiwu

    2016-01-01

    The Z mutation (E342K) of α1-antitrypsin (α1-AT), carried by 4% of Northern Europeans, predisposes to early onset of emphysema due to decreased functional α1-AT in the lung and to liver cirrhosis due to accumulation of polymers in hepatocytes. However, it remains unclear why the Z mutation causes intracellular polymerization of nascent Z α1-AT and why 15% of the expressed Z α1-AT is secreted into circulation as functional, but polymerogenic, monomers. Here, we solve the crystal structure of the Z-monomer and have engineered replacements to assess the conformational role of residue Glu-342 in α1-AT. The results reveal that Z α1-AT has a labile strand 5 of the central β-sheet A (s5A) with a consequent equilibrium between a native inhibitory conformation, as in its crystal structure here, and an aberrant conformation with s5A only partially incorporated into the central β-sheet. This aberrant conformation, induced by the loss of interactions from the Glu-342 side chain, explains why Z α1-AT is prone to polymerization and readily binds to a 6-mer peptide, and it supports that annealing of s5A into the central β-sheet is a crucial step in the serpins' metastable conformational formation. The demonstration that the aberrant conformation can be rectified through stabilization of the labile s5A by binding of a small molecule opens a potential therapeutic approach for Z α1-AT deficiency. PMID:27246852

  18. 21 CFR 866.5580 - Alpha-1-lipoprotein immuno-logical test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alpha-1-lipoprotein immuno-logical test system. 866.5580 Section 866.5580 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN....5580 Alpha-1-lipoprotein immuno-logical test system. (a) Identification. An...

  19. Antagonism of Lateral Amygdala Alpha1-Adrenergic Receptors Facilitates Fear Conditioning and Long-Term Potentiation

    ERIC Educational Resources Information Center

    Lazzaro, Stephanie C.; Hou, Mian; Cunha, Catarina; LeDoux, Joseph E.; Cain, Christopher K.

    2010-01-01

    Norepinephrine receptors have been studied in emotion, memory, and attention. However, the role of alpha1-adrenergic receptors in fear conditioning, a major model of emotional learning, is poorly understood. We examined the effect of terazosin, an alpha1-adrenergic receptor antagonist, on cued fear conditioning. Systemic or intra-lateral amygdala…

  20. The degree of branching in (alpha 1,4)-(alpha 1,6)-linked glucopolysaccharides is dependent on intrinsic properties of the branching enzymes.

    PubMed

    Tolmasky, D S; Krisman, C R

    1987-10-15

    1. Branching enzymes from rat and rabbit liver, as well as from potato and maize were prepared. They were almost free from contaminating glucan-degrading enzymes. 2. In 'sweet corn' maize, two separate fractions with (alpha 1,4)glucan: (alpha 1,4)glucan alpha 6-glycosyltransferase activities were obtained. One of them synthesized amylopectin, the branched component of starch, in the presence of phosphorylase and Glc1P, while the other fraction synthesized phytoglycogen. Furthermore, in a maize variety which does not accumulate phytoglycogen, only one fraction of branching activity was found, that formed amylopectin under the above-mentioned conditions. 3. Comparative analyses performed with native (alpha 1,4)-(alpha 1,6)glucopolysaccharides, and those synthesized in vitro with the branching enzyme from the same tissue, demonstrated a close similarity between both glucans. 4. It may be concluded that the branching enzyme is responsible for the specific degree of (alpha 1,6) branch linkages found in the native polysaccharide. PMID:2959476

  1. The degree of branching in (alpha 1,4)-(alpha 1,6)-linked glucopolysaccharides is dependent on intrinsic properties of the branching enzymes.

    PubMed

    Tolmasky, D S; Krisman, C R

    1987-10-15

    1. Branching enzymes from rat and rabbit liver, as well as from potato and maize were prepared. They were almost free from contaminating glucan-degrading enzymes. 2. In 'sweet corn' maize, two separate fractions with (alpha 1,4)glucan: (alpha 1,4)glucan alpha 6-glycosyltransferase activities were obtained. One of them synthesized amylopectin, the branched component of starch, in the presence of phosphorylase and Glc1P, while the other fraction synthesized phytoglycogen. Furthermore, in a maize variety which does not accumulate phytoglycogen, only one fraction of branching activity was found, that formed amylopectin under the above-mentioned conditions. 3. Comparative analyses performed with native (alpha 1,4)-(alpha 1,6)glucopolysaccharides, and those synthesized in vitro with the branching enzyme from the same tissue, demonstrated a close similarity between both glucans. 4. It may be concluded that the branching enzyme is responsible for the specific degree of (alpha 1,6) branch linkages found in the native polysaccharide.

  2. INHALED ALPHA1-PROTEINASE INHIBITOR THERAPY IN PATIENTS WITH CYSTIC FIBROSIS

    PubMed Central

    Gaggar, Amit; Chen, Junliang; Chmiel, James F; Dorkin, Henry L; Flume, Patrick A; Griffin, Rhonda; Nichols, David; Donaldson, Scott H

    2016-01-01

    Background Inhaled alpha1-proteinase inhibitor (PI) is known to reduce neutrophil elastase burden in some patients with CF. This phase 2a study was designed to test inhaled Alpha-1 HC, a new aerosolized alpha1-PI formulation, in CF patients. Methods We performed a randomized, double-blind, placebo-controlled study and evaluated the safety of 100 or 200 mg of inhaled Alpha-1 HC once daily for 3 weeks in subjects with CF. Thirty adult subjects were randomized in a 2:1 ratio to receive Alpha-1 HC or placebo. Results Drug delivery was confirmed by a dose-dependent increase in the sputum alpha1-PI. Seven (20.0%) of the 35 adverse events in the 100-mg dose group, 3 (13.0%) of 23 in the 200-mg dose group, and 4 (14.3%) of 28 in the placebo group were drug-related in these subjects. One serious adverse event occurred in 1 subject within each group. Conclusions Alpha-1 HC inhalation was safe and well tolerated. PMID:26321218

  3. Contrasting signaling pathways of alpha1A- and alpha1B-adrenergic receptor subtype activation of phosphatidylinositol 3-kinase and Ras in transfected NIH3T3 cells.

    PubMed

    Hu, Z W; Shi, X Y; Lin, R Z; Hoffman, B B

    1999-01-01

    Activation of protein kinases is an important intermediate step in signaling pathways of many G protein-coupled receptors including alpha1-adrenergic receptors. The present study was designed to investigate the capacity of the three cloned subtypes of human alpha1-receptors, namely, alpha1A, alpha1B and alpha1D to activate phosphatidylinositol 3-kinase (PI 3-kinase) and p21ras in transfected NIH3T3 cells. Norepinephrine activated PI 3-kinase in cells expressing human alpha1A and alpha1B via pertussis toxin-insensitive G proteins; alpha1D-receptors did not detectably activate this kinase. Transient transfection of NIH 3T3 cells with the alpha-subunit of the G protein transducin (alpha(t)) a scavenger of betagamma-subunits released from activated G proteins, inhibited alpha1B-receptor but not alpha1A-receptor-stimulated PI 3-kinase activity. Stimulation of both alpha1A- and alpha1B-receptors activated p21ras and stimulated guanine nucleotide exchange on Ras protein. Overexpression of a dominant negative mutant of p21ras attenuated alpha1B-receptor but not alpha1A-receptor activation of PI 3-kinase. Overexpression of a dominant negative mutant of PI 3-kinase attenuated alpha1A- but not alpha1B-receptor-stimulated mitogen-activated protein kinase activity. These results demonstrate the capacity for heterologous signaling of the alpha1-adrenergic receptor subtypes in promoting cellular responses in NIH3T3 cells.

  4. Secretion of high-mannose-type alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein by primary cultures of rat hepatocytes in the presence of the mannosidase I inhibitor 1-deoxymannojirimycin.

    PubMed

    Gross, V; Steube, K; Tran-Thi, T A; McDowell, W; Schwarz, R T; Decker, K; Gerok, W; Heinrich, P C

    1985-07-01

    Two different forms of alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein were found in primary cultures of rat hepatocytes. After a 2.5-h labeling period with [35S]methionine the high-mannose-type precursor of alpha 1-proteinase inhibitor (Mr 49000) and alpha 1-acid glycoprotein (Mr 39 000) and the mature-complex-type alpha 1-proteinase inhibitor (Mr 54 000) and alpha 1-acid glycoprotein (Mr 43 000-60 000) could be immunoprecipitated from the cells, but only the complex-type forms of the two glycoproteins were secreted into the hepatocyte media. When hepatocytes were incubated with the mannosidase I inhibitor 1-deoxymannojirimycin at a concentration of 4 mM, the 49 000-Mr form of alpha 1-proteinase inhibitor and the 39 000-Mr form of alpha 1-acid glycoprotein could be detected in the cells as well as in their media. Neither the secretion of alpha 1-proteinase inhibitor nor that of alpha 1-acid glycoprotein was impaired by 1-deoxymannojirimycin. While alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein, secreted by control cells, were resistant to endoglucosaminidase H, alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein, secreted by hepatocytes treated with 4 mM 1-deoxymannojirimycin, could be deglycosylated by endoglucosaminidase H. When the [3H]mannose-labeled oligosaccharides of alpha 1-proteinase inhibitor, secreted by 1-deoxymannojirimycin-treated hepatocytes, were cleaved off by endoglucosaminidase H and analyzed by Bio-Gel P-4 chromatography, they eluted at the position of Man9GlcNAc, indicating that mannosidase I had been efficiently inhibited. 1-Deoxymannojirimycin did not inhibit the synthesis or the cotranslational N-glycosylation of alpha 1-proteinase inhibitor or alpha 1-acid glycoprotein. PMID:3160588

  5. Overexpression of laminin alpha1 chain in colonic cancer cells induces an increase in tumor growth.

    PubMed

    De Arcangelis, A; Lefebvre, O; Méchine-Neuville, A; Arnold, C; Klein, A; Rémy, L; Kedinger, M; Simon-Assmann, P

    2001-10-01

    Laminins represent a growing family of glycoproteins constituting the basement membrane. They are known to direct many biological processes. With respect to carcinogenesis, laminins play an important role in cell adhesion, mitogenesis, differentiation and even metastasis. To further study the biological significance of laminin-1 (composed of alpha1, beta1 and gamma1 chains) in intestinal cell differentiation or tumorigenesis, an alpha1-laminin expression vector was introduced into the HT29 colonic cancer cells, in which laminin alpha1 chain is not expressed. Upon transfection of the alpha1 chain, the alpha1beta1gamma1 trimer was found secreted in the media along with free alpha1 chain as assessed by immunoprecipitation. The presence of the laminin alpha1 chain did not significantly modify the levels of the other laminin chains nor the integrins expressed by the HT29 cells. In spite of similar growth properties with the control cells in vitro (plastic dish, soft agar), the laminin alpha1 transfectants showed a significantly increased tumor growth when injected in nude mice. Histologic and immunohistochemic examination of the laminin alpha1-expressing tumors points to an increased recruitment of the host stromal and vascular cells, without modification in the differentiation profile and invasion potential. In parallel, a clear accumulation of laminin-10 (alpha5beta1gamma1) at the carcinoma/stromal interface and a segregation of the integrin beta4 subunit at the basal pole of the cancer cells occurred, compared to control tumors. Overall, our observations emphasize the importance of laminin-1 as a chemoattractant of both stromal and vascular cells and in epithelial/stromal cell interactions for the organization of the basement membrane and segregation of integrins leading to an epithelial cell growth signal. Such a sequence of events is reminiscent of what occurs during development.

  6. Physical mapping of four serpin genes: [alpha][sub 1]-antitrypsin, [alpha][sub 1]-antichymotrypsin, corticosteroid-binding globulin, and protein C inhibitor, within a 280-kb region on chromosome 14q32. 1

    SciTech Connect

    Billingsley, G.D.; Cox, D.W. Univ. of Toronto, Ontario ); Walter, M.A. ); Hammond, G.L. )

    1993-02-01

    Alpha[sub 1]-antitrypsin ([alpha]1AT; protease inhibitor [PI] locus), [alpha][sub 1]-antichymotrypsin ([alpha]1ACT; AACT locus), corticosteroid-binding globulin (CBG; CBG locus), and protein C inhibitor (PCI; PCI locus) are members of the serine protease inhibitor (serpin) superfamily. A noncoding PI-like (PIL) gene has been located 12 kb 3[prime] of the PI gene. The PI, PIL, and AACT loci have been localized to 14q32.1, the CBG locus has been localized to 14q31-14q32.1, and PCI has been mapped to chromosome 14. Genetic linkage analysis suggests tight linkage between PI and AACT. The authors have used pulsed-field gel electrophoresis to generate a physical map linking these five serpin genes. The order of the genetic loci is AACT/PCI-PI-PIL-CBG, with a maximum distance of about 220 kb between the AACT/PCI and PI genes. These genes form a PI cluster at 14q32.1, similar to that of the homologous genes on murine chromosome 12. The close proximity of these genes has implications for disease-association studies. 44 refs., 6 figs., 2 tabs.

  7. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity

    PubMed Central

    Ordóñez, Adriana; Pérez, Juan; Tan, Lu; Dickens, Jennifer A.; Motamedi-Shad, Neda; Irving, James A.; Haq, Imran; Ekeowa, Ugo; Marciniak, Stefan J.; Miranda, Elena; Lomas, David A.

    2015-01-01

    Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin.—Ordóñez, A., Pérez, J., Tan, L., Dickens, J. A., Motamedi-Shad, N., Irving, J. A., Haq, I., Ekeowa, U., Marciniak, S. J., Miranda, E., Lomas, D. A. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity. PMID:25757566

  8. Association between alphas1-, beta- and kappa-casein loci in two Italian cattle breeds.

    PubMed

    Voglino, G F; Carignano

    1975-01-01

    The genetic polymorphism alphas1-, beta- and kappa-caseins was examined by gel electrophoresis in two Italian breeds, Valdostana and Piedmont. The results obtained from acid and basic migration show that the gene frequencies of the two breeds are very similar. Non independent assortment of genotypes among these milk protein loci was also studied. Results of analyses carried out on loci pairs showed that the genetic complex alphas1-CnB - beta-CnA2 was the most common in both breeds. In addition, the measure of linkage disequilibrium or gametic association (denoted delta) showed a close association between alphas1-Cn and beta-Cn, and between beta-Cn and kappa-Cn. No significant association was found between alphas1-Cn and kappa-Cn. This is in line with the model proposed by Grosclaude et al. (1973).

  9. Estrogen alters the diurnal rhythm of alpha 1-adrenergic receptor densities in selected brain regions

    SciTech Connect

    Weiland, N.G.; Wise, P.M.

    1987-11-01

    Norepinephrine regulates the proestrous and estradiol-induced LH surge by binding to alpha 1-adrenergic receptors. The density of alpha 1-receptors may be regulated by estradiol, photoperiod, and noradrenergic neuronal activity. We wished to determine whether alpha 1-receptors exhibit a diurnal rhythm in ovariectomized and/or estradiol-treated female rats, whether estradiol regulates alpha 1-receptors in those areas of brain involved with LH secretion and/or sexual behavior, and whether the concentrations of alpha-receptors vary inversely relative to previously reported norepinephrine turnover patterns. Young female rats, maintained on a 14:10 light-dark cycle were ovariectomized. One week later, half of them were outfitted sc with Silastic capsules containing estradiol. Groups of animals were decapitated 2 days later at 0300, 1000, 1300, 1500, 1800, and 2300 h. Brains were removed, frozen, and sectioned at 20 micron. Sections were incubated with (/sup 3/H)prazosin in Tris-HCl buffer, washed, dried, and exposed to LKB Ultrofilm. The densities of alpha 1-receptors were quantitated using a computerized image analysis system. In ovariectomized rats, the density of alpha 1-receptors exhibited a diurnal rhythm in the suprachiasmatic nucleus (SCN), medial preoptic nucleus (MPN), and pineal gland. In SCN and MPN, receptor concentrations were lowest during the middle of the day and rose to peak levels at 1800 h. In the pineal gland, the density of alpha 1-receptors was lowest at middark phase, rose to peak levels before lights on, and remained elevated during the day. Estradiol suppressed the density of alpha 1 binding sites in the SCN, MPN, median eminence, ventromedial nucleus, and the pineal gland but had no effect on the lateral septum. Estrogen treatment altered the rhythm of receptor densities in MPN, median eminence, and the pineal gland.

  10. Phorbol esters promote alpha 1-adrenergic receptor phosphorylation and receptor uncoupling from inositol phospholipid metabolism.

    PubMed Central

    Leeb-Lundberg, L M; Cotecchia, S; Lomasney, J W; DeBernardis, J F; Lefkowitz, R J; Caron, M G

    1985-01-01

    DDT1 MF-2 cells, which are derived from hamster vas deferens smooth muscle, contain alpha 1-adrenergic receptors (54,800 +/- 2700 sites per cell) that are coupled to stimulation of inositol phospholipid metabolism. Incubation of these cells with tumor-promoting phorbol esters, which stimulate calcium- and phospholipid-dependent protein kinase, leads to a marked attenuation of the ability of alpha 1-receptor agonists such as norepinephrine to stimulate the turnover of inositol phospholipids. This turnover was measured by determining the 32P content of phosphatidylinositol and phosphatidic acid after prelabeling of the cellular ATP pool with 32Pi. These phorbol ester-treated cells also displayed a decrease in binding affinity of cellular alpha 1 receptors for agonists with no change in antagonist affinity. By using affinity chromatography on the affinity resin Affi-Gel-A55414, the alpha 1 receptors were purified approximately equal to 300-fold from control and phorbol ester-treated 32Pi-prelabeled cells. As assessed by NaDodSO4/polyacrylamide gel electrophoresis, the Mr 80,000 alpha 1-receptor ligand-binding subunit is a phosphopeptide containing 1.2 mol of phosphate per mol of alpha 1 receptor. After phorbol ester treatment this increased to 3.6 mol of phosphate per mol of alpha 1 receptor. The effect of phorbol esters on norepinephrine-stimulated inositol phospholipid turnover and alpha 1-receptor phosphorylation showed the same rapid time course with a t1/2 less than 2 min. These results indicate that calcium- and phospholipid-dependent protein kinase may play an important role in regulating the function of receptors that are coupled to the inositol phospholipid cycle by phosphorylating and deactivating them. Images PMID:2994039

  11. Isolation of alpha 1-protease inhibitor from human normal and malignant ovarian tissue.

    PubMed Central

    Bagdasarian, A; Wheeler, J; Stewart, G J; Ahmed, S S; Colman, R W

    1981-01-01

    Proteolytic enzymes are associated with normal and neoplastic tissues. Therefore protease inhibitors might also be involved in the control of cell function. alpha 1-protease antigen and antitryptic activity have been found in normal and neoplastic human ovarian homogenate. The inhibitor has been localized to ovarian stromal cells or tumor cells by immunoperoxidase staining. The protein was purified to apparent homogeneity as judged by alkaline gel and sodium dodecyl sulfate (SDS) gel electrophoresis. Immunochemical studies revealed antigenic similarity of plasma alpha 1-protease inhibitor by double immunodiffusion and similar mobility on immunoelectrophoresis and two-dimensional electroimmunodiffusion. The molecular weight was similar to that described for plasma alpha 1-protease inhibitor: 60,000 by gel filtration and 53,500 by SDS electrophoresis. Furthermore, the phenotypic pattern as determined by acid starch gel electrophoresis and immunoprecipitation was PiMM, which is the predominant genetic variant in normal plasma alpha 1-protease inhibitor. An inhibitor ws isolated and purified from an ovarian carcinoma that exhibited functional, immunochemical, and physical similarity to the normal ovarian alpha 1-protease inhibitor. alpha 1-protease inhibitor from normal and malignant ovaries competitively inhibited bovine pancreatic trypsin at incubation times of 5 min at 30 degrees C. Inhibition constant (Ki) values were calculated at 0.67 and 0.51 inhibitory units, respectively. The alpha 1-protease inhibitor in malignant cells may be a factor in the control of proliferation in this tissue. Since ovulation is in part a proteolytic event, the alpha 1-protease inhibitor in ovarian cells may play a role in the control of this specialized tissue. Persistance of this protein in malignant ovarian tissue may be a vestige of its differentiated origin. Images PMID:6161137

  12. Effect of extracellular pH on recombinant alpha1beta2gamma2 and alpha1beta2 GABAA receptors.

    PubMed

    Mercik, Katarzyna; Pytel, Maria; Cherubini, Enrico; Mozrzymas, Jerzy W

    2006-08-01

    Recently, we have reported that extracellular protons allosterically modulated neuronal GABA(A) receptors [Mozrzymas, J.W., Zarnowska, E.D., Pytel, M., Mercik, K., 2003a. Modulation of GABA(A) receptors by hydrogen ions reveals synaptic GABA transient and a crucial role of desensitiztion process. Journal of Neuroscience 23, 7981-7992]. However, GABAARs in neurons are heterogeneous and the effect of hydrogen ions depends on the receptor subtype. In particular, gamma2 subunit sets the receptor sensibility to several modulators including protons. However, the mechanisms whereby protons modulate gamma2-containing and gamma2-free GABAARs have not been fully elucidated. To this end, current responses to ultrafast GABA applications were recorded for alpha1beta2gamma2 and alpha1beta2 receptors at different pH values. For both receptor types, increase in pH induced a decrease in amplitudes of currents elicited by saturating [GABA] but this effect was stronger for alpha1beta2 receptors. In the case of alpha1beta2gamma2 receptors, protons strongly affected the current time course due to a down regulation of binding and desensitization rates. This effect was qualitatively similar to that described in neurons. Protons strongly influenced the amplitude of alpha1beta2 receptor-mediated currents but the effect on their kinetics was weak suggesting a predominant direct non-competitive inhibition with a minor allosteric modulation. In conclusion, we provide evidence that extracellular protons strongly affect GABAA receptors and that, depending on the presence of the gamma2 subunit, the modulatory mechanisms show profound quantitative and qualitative differences.

  13. Postnatal development of the alpha1 containing GABAA receptor subunit in rat hippocampus.

    PubMed

    Lopez-Tellez, Juan Felix; Vela, Jose; del Rio, Juan Carlos; Ramos, Blanca; Baglietto-Vargas, David; Santa-Maria, Consuelo; Ruano, Diego; Gutierrez, Antonia; Vitorica, Javier

    2004-01-31

    Here we have studied the developmental expression of alpha1 subunit of the GABAA receptor in comparison with the expression of alpha2 subunit and several GABAergic markers (parvalbumin (PV), calretinin (CR), somatostatin (SOM), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP)). The alpha1 expression (mRNA and protein) was low at birth and increased progressively until the adulthood. This expression pattern was similar to that observed for PV, opposite to that of CR (high at birth and decreased continuously until the adulthood) and differed from that observed for the alpha2 and neuropeptides (SOM, NPY and VIP) (in all cases, a clear peak in expression was observed at P10). We further investigated the expression of alpha1, PV and CR by immunohistochemistry. As expected, the alpha1 and the PV expression were low at birth and increased progressively until the adulthood. Both alpha1 and PV were co-expressed by the same interneuronal population, however, the maturation of the alpha1 subunit preceded to that of PV. Finally, we observed a gradient of maturation between the different fields of the hippocampus proper (CA2-3 preceded to CA1 and DG). This gradient could be related to the high expression of CR positive cells and fibers during the first 10 postnatal days, located principally in the stratum lacunosum moleculare of the CA2-3 layers.

  14. Synthesis of the core tetrasaccharide of Trypanosoma cruzi glycoinositolphospholipids: Manp(alpha1-->6)-Manp(alpha1-->4)-6-(2-aminoethylphosphonic acid)-GlcNp(alpha1-->6)-myo-Ins-1-PO4.

    PubMed

    Hederos, Markus; Konradsson, Peter

    2005-09-01

    [structure: see text] Synthesis of the core tetrasaccharide Manp(alpha1-->6)-Manp(alpha1-->4)-6-(2-aminoethylphosphonic acid)-GlcNp(alpha1-->6)-myo-Ins-1-PO4, found in glycoinositolphospholipids of Trypanosoma cruzi parasites, is described. The key building block, 6-O-(2-azido-3-O-benzyl-6-O-((2-benzyloxycarbonylaminoethyl)phosphonic acid benzyl ester)-2-deoxy-alpha-D-glucopyranosyl)-1-di-O-benzylphosphoryl-4,5-O-isopropylidene-2,3-O-(D-1,7,7-trimethyl[2,2,1]bicyclohept-6-ylidene)-D-myo-inositol, was synthesized using a partially protected glucosyl D-camphorinositolphosphate and a (2-benzyloxycarbonylaminoethyl)phosphonic acid derivative in a regioselective phosphonate esterfication. Elongation with ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzyl-1-alpha-D-thiomannopyranoside using dimethyl(methylthio)sulfonium trifluoromethanesulfonate gave a fully protected tetrasaccharide which was successfully deprotected subsequently with sodium methoxide, sodium in liquid ammonia, and aq hydrochloric acid to give title compound.

  15. Expression of human. alpha. sub 1 -antitrypsin in dogs after autologous transplantation of retroviral transduced hepatocytes

    SciTech Connect

    Kay, M.A.; Baley, P.; Rothenberg, S.; Leland, F; Fleming, L.; Ponder, K.P.; Liu, Tajen; Finegold, M.; Darlington, G.; Pokorny, W.; Woo, S.L.C. )

    1992-01-01

    The liver represents an excellent organ for gene therapy since many genetic disorders result from the deficiency of liver-specific gene products. The authors have previously demonstrated that transgenic mouse hepatocytes can be heterologously transplanted into congenic recipients where they survived indefinitely and continued to function as hepatocytes. Here they demonstrate the autologous transplantation of retrovirally transduced canine hepatocytes. In two animals they have transplanted hepatocytes transduced with a retroviral vector containing the human {alpha}{sub 1}-antitrypsin cDNA under transcriptional control of the cytomegalovirus promotor. Both animals had significant human {alpha}{sub 1}-antitrypsin in the serum for 1 month. The results suggest that gene therapy of hepatic deficiencies may be achieved by hepatocellular transplantation after genetic reconstruction with the use of promoters of cellular genes that are active in the normal liver.

  16. Framework for Interpretation of Trypsin–antitrypsin Imbalance and Genetic Heterogeneity in Pancreatitis

    PubMed Central

    Lin, Kun; Gao, Feng; Chen, Qingquan; Liu, Qicai; Chen, Shu

    2015-01-01

    Early intracellular premature trypsinogen activation was interpreted as the key initiator of pancreatitis. When the balance in the homeostasis of trypsin and antitrypsin system is disequilibrated, elevated aggressive enzymes directly attack the pancreatic tissue, which leads to pancreatic destruction and inflammation. However, trypsin alone is not enough to cause complications in pancreatitis, which may play a crucial role in modulating signaling events in the initial phase of the disease. NFκB activation is the major inflammatory pathway involved in the occurrence and development of pancreatitis and it can be induced by intrapancreatic activation of trypsinogen. Synthesis of trypsinogen occurs in endoplasmic reticulum (ER), and ER stress is an important early acinar cell event. Components of ER stress response are known to be able to trigger cell death as well as NFκB signaling cascade. The strongest evidence supporting the trypsin-centered theory is that gene mutations, which lead to the generation of more trypsin, or reduce the activity of trypsin inhibitors or trypsin degradation, are associated with pancreatitis. Thus, trypsin–antitrypsin imbalance may be the first step leading to pancreatic autodigestion and inducing other pathways. Continued experimental studies are necessary to determine the specific relationships between trypsin–antitrypsin imbalance and genetic heterogeneity in pancreatitis. In this article, we review the latest advances that contributed to the understanding of the basic mechanisms behind the occurrence and development of pancreatitis with a focus on the interpretation of trypsin–antitrypsin imbalance and their relationships with other inflammation pathways. We additionally highlight genetic predispositions to pancreatitis and possible mechanisms associated with them. PMID:26228362

  17. Comparison of the antagonistic activity of tamsulosin and doxazosin at vascular alpha 1-adrenoceptors in humans.

    PubMed

    Harada, K; Ohmori, M; Fujimura, A

    1996-11-01

    alpha 1-Adrenoceptor blockers such as prazosin and doxazosin are used to treat hypertension as well as benign prostatic hyperplasia (BPH), whereas the new alpha 1-adrenoceptor blocker tamsulosin is used only for BPH and does not reduce blood pressure at the doses used to relax prostatic smooth muscle. In contrast to prazosin, tamsulosin has a higher affinity for prostatic than vascular alpha 1-adrenoceptors in vitro. The functional correlate of this observation in humans is the subject of this study. The alpha 1-adrenoceptor blockade by oral tamsulosin (0.2 mg), doxazosin (1 mg) or placebo on finger tip vascular and dorsal hand venous alpha 1-adrenoceptors stimulated by cold treatment (immersion in ice water) and the alpha 1-adrenoceptor agonist phenylephrine, was thus studied in a 3-way crossover study in eight, healthy, male adults. Finger tip vasoconstriction after cold stimulation was assessed by laser Doppler flowmetry. A linear variable differential transformer was used to assess the drug effect on phenylephrine-induced venoconstriction. All study parameters were assessed at around 2 and 3.5 h after oral intake of doxazosin and tamsulosin respectively. The drug plasma levels were not significantly different. No significant differences were found for blood pressure or heart rate in the three treatments in supine and erect position. The reduction in finger tip blood flow after cold stimulation was significantly smaller after doxazosin treatment (P < 0.01) than after tamsulosin or placebo, whereas there was no significant difference between tamsulosin and placebo treatments. The infusion rate of phenylephrine producing a half-maximum venoconstriction was significantly larger after doxazosin than after tamsulosin (P < 0.05) or placebo (P < 0.01), whereas there was again no significant difference between tamsulosin and placebo treatments. The data suggest that, at doses producing equal plasma levels after single oral doses in human subjects, the blocking activity

  18. Accelerated telomere attrition in children and teenagers with α1-antitrypsin deficiency.

    PubMed

    Escribano, Amparo; Pastor, Sara; Reula, Ana; Castillo, Silvia; Vicente, Silvia; Sanz, Francisco; Casas, Francisco; Torres, María; Fernández-Fabrellas, Estrella; Codoñer-Franch, Pilar; Dasí, Francisco

    2016-08-01

    Numerous studies have shown that oxidative stress accelerates telomere shortening in several lung pathologies. Since oxidative stress is involved in the pathophysiology of α1-antitrypsin deficiency (AATD), we hypothesised that telomere shortening would be accelerated in AATD patients. This study aimed to assess telomere length in AATD patients and to study its association with α1-antitrypsin phenotypes.Telomere length, telomerase activity, telomerase reverse transcriptase (hTERT) expression and biomarkers of oxidative stress were measured in 62 children and teenagers (aged 2-18 years) diagnosed with AATD and 18 controls (aged 3-16 years).Our results show that intermediate-risk (MZ; SZ) and high-risk (ZZ) AATD patients have significantly shorter telomeres and increased oxidative stress than controls. Correlation studies indicate that telomere length was related to oxidative stress markers in AATD patients. Multiple hypothesis testing revealed an association between telomere length, telomerase activity, hTERT expression and AATD phenotypes; high-risk patients showed shorter telomeres, lower hTERT expression and decreased telomerase activity than intermediate-risk and low-risk patients.AATD patients show evidence of increased oxidative stress leading to telomere attrition. An association between telomere and α1-antitrypsin phenotypes is observed suggesting that telomere length could be a promising biomarker for AATD disease progression.

  19. Ca(2+)-dependent inactivation of a cloned cardiac Ca2+ channel alpha 1 subunit (alpha 1C) expressed in Xenopus oocytes.

    PubMed Central

    Neely, A; Olcese, R; Wei, X; Birnbaumer, L; Stefani, E

    1994-01-01

    The alpha 1 subunit of cardiac Ca2+ channel, expressed alone or coexpressed with the corresponding beta subunit in Xenopus laevis oocytes, elicits rapidly inactivating Ca2+ currents. The inactivation has the following properties: 1) It is practically absent in external Ba2+; 2) it increases with Ca2+ current amplitudes; 3) it is faster at more negative potentials for comparable Ca2+ current amplitudes; 4) it is independent of channel density; and 5) it does not require the beta subunit. These findings indicate that the Ca2+ binding site responsible for inactivation is encoded in the alpha 1 subunit and suggest that it is located near the inner channel mouth but outside the membrane electric field. PMID:8075326

  20. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

    PubMed

    Ordóñez, Adriana; Pérez, Juan; Tan, Lu; Dickens, Jennifer A; Motamedi-Shad, Neda; Irving, James A; Haq, Imran; Ekeowa, Ugo; Marciniak, Stefan J; Miranda, Elena; Lomas, David A

    2015-06-01

    Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin. PMID:25757566

  1. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

    PubMed

    Ordóñez, Adriana; Pérez, Juan; Tan, Lu; Dickens, Jennifer A; Motamedi-Shad, Neda; Irving, James A; Haq, Imran; Ekeowa, Ugo; Marciniak, Stefan J; Miranda, Elena; Lomas, David A

    2015-06-01

    Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin.

  2. Posttranscriptional regulation of collagen alpha1(I) mRNA in hepatic stellate cells.

    PubMed Central

    Stefanovic, B; Hellerbrand, C; Holcik, M; Briendl, M; Aliebhaber, S; Brenner, D A

    1997-01-01

    The hepatic stellate cell (HSC) is the primary cell responsible for the dramatic increase in the synthesis of type I collagen in the cirrhotic liver. Quiescent HSCs contain a low level of collagen alpha1(I) mRNA, while activated HSCs contain about 60- to 70-fold more of this mRNA. The transcription rate of the collagen alpha1(I) gene is only two fold higher in activated HSCs than in quiescent HSCs. In assays using actinomycin D or 5,6-dichlorobenzimidazole riboside collagen alpha1(I) mRNA has estimated half-lives of 1.5 h in quiescent HSCs and 24 h in activated HSCs. Thus, this 16-fold change in mRNA stability is primarily responsible for the increase in collagen alpha1(I) mRNA steady-state level in activated HSCs. We have identified a novel RNA-protein interaction targeted to the C-rich sequence in the collagen alpha1(I) mRNA 3' untranslated region (UTR). This sequence is localized 24 nucleotides 3' to the stop codon. In transient transfection experiments, mutation of this sequence diminished accumulation of an mRNA transcribed from a collagen alpha1(I) minigene and in stable transfections decreased the half-life of collagen alpha1(I) minigene mRNA. Binding to the collagen alpha1(I) 3' UTR is present in cytoplasmic extracts of activated but not quiescent HSCs. It contains as a subunit alphaCP, which is also found in the complex involved in stabilization of alpha-globin mRNA. The auxiliary factors necessary to promote binding of alphaCP to the collagen 3' UTR are distinct from the factors necessary for binding to the alpha-globin sequence. Since alphaCP is expressed in both quiescent and activated HSCs, these auxiliary factors are responsible for the differentially expressed RNA-protein interaction at the collagen alpha1(I) mRNA 3' UTR. PMID:9271398

  3. DNA elements regulating alpha1-tubulin gene induction during regeneration of eukaryotic flagella.

    PubMed

    Periz, G; Keller, L R

    1997-07-01

    Eukaryotic flagella are complex organelles composed of more than 200 polypeptides. Little is known about the regulatory mechanisms governing synthesis of the flagellar protein subunits and their assembly into this complex organelle. The unicellular green alga Chlamydomonas reinhardtii is the premier experimental model system for studying such cellular processes. When acid shocked, C. reinhardtii excises its flagella, rapidly and coordinately activates transcription of a set of flagellar genes, and ultimately regenerates a new flagellar pair. To define functionally the regulatory sequences that govern induction of the set of genes after acid shock, we analyzed the alpha1-tubulin gene promoter. To simplify transcriptional analysis in vivo, we inserted the selectable marker gene ARG7 on the same plasmid with a tagged alpha1-tubulin gene and stably introduced it into C. reinhardtii cells. By deletion of various sequences, two promoter regions (-176 to -122 and -85 to -16) were identified as important for induction of the tagged alpha1-tubulin gene. Deleting the region between -176 and -122 from the transcription start site resulted in an induction level which was only 45 to 70% of that of the resident gene. Deleting the region upstream of -56 resulted in a complete loss of inducibility without affecting basal expression. The alpha1-tubulin promoter region from -85 to -16 conferred partial acid shock inducibility to an arylsulfatase (ARS) reporter gene. These results show that induction of the alpha1-tubulin gene after acid shock is a complex response that requires diverse sequence elements.

  4. DNA elements regulating alpha1-tubulin gene induction during regeneration of eukaryotic flagella.

    PubMed Central

    Periz, G; Keller, L R

    1997-01-01

    Eukaryotic flagella are complex organelles composed of more than 200 polypeptides. Little is known about the regulatory mechanisms governing synthesis of the flagellar protein subunits and their assembly into this complex organelle. The unicellular green alga Chlamydomonas reinhardtii is the premier experimental model system for studying such cellular processes. When acid shocked, C. reinhardtii excises its flagella, rapidly and coordinately activates transcription of a set of flagellar genes, and ultimately regenerates a new flagellar pair. To define functionally the regulatory sequences that govern induction of the set of genes after acid shock, we analyzed the alpha1-tubulin gene promoter. To simplify transcriptional analysis in vivo, we inserted the selectable marker gene ARG7 on the same plasmid with a tagged alpha1-tubulin gene and stably introduced it into C. reinhardtii cells. By deletion of various sequences, two promoter regions (-176 to -122 and -85 to -16) were identified as important for induction of the tagged alpha1-tubulin gene. Deleting the region between -176 and -122 from the transcription start site resulted in an induction level which was only 45 to 70% of that of the resident gene. Deleting the region upstream of -56 resulted in a complete loss of inducibility without affecting basal expression. The alpha1-tubulin promoter region from -85 to -16 conferred partial acid shock inducibility to an arylsulfatase (ARS) reporter gene. These results show that induction of the alpha1-tubulin gene after acid shock is a complex response that requires diverse sequence elements. PMID:9199320

  5. Effect of repeated treatment with tianeptine and fluoxetine on the central alpha(1)-adrenergic system.

    PubMed

    Rogóz, Z; Skuza, G; Dlaboga, D; Maj, J; Dziedzicka-Wasylewska, M

    2001-09-01

    Tianeptine (TIA) is an antidepressant drug which enhances the reuptake of serotonin but, in contrast to tricyclics, shows no affinity for neurotransmitter receptors. The present study was aimed at determining whether repeated TIA treatment induced adaptive changes in the alpha(1)-adrenergic system, similar to those reported by us earlier for tricyclic antidepressants. The experiments were carried out on male mice and rats. TIA was administered at a dose of 5 or 10mg/kg once or repeatedly (twice daily for 14 days) and fluoxetine (FLU), used as a reference compound, at a dose of 10mg/kg. The obtained results showed that TIA administered repeatedly potentiated the methoxamine- and phenylephrine (PHEN)-induced exploratory hyperactivity in rats and clonidine-induced aggressiveness in mice, the effects mediated by alpha(1)-adrenoceptors. TIA given repeatedly (but not acutely) increased the binding (B(max)) of alpha(1)-adrenergic receptors in cerebral cortex for [(3)H]prazosin. However, the ability of the alpha(1)-adrenoceptor agonist PHEN to compete for these sites was not significantly changed. The above results indicate that repeated TIA administration increases the responsiveness of the alpha(1)-adrenergic system (behavioural and biochemical changes). On the other hand, FLU did not affect any behavioural and biochemical changes in this system.

  6. Effect of alpha 1-adrenoceptor blockade on resting and hyperemic myocardial blood flow in normal humans.

    PubMed

    Lorenzoni, R; Rosen, S D; Camici, P G

    1996-10-01

    In the present study we aimed to assess the effect of alpha 1-adrenoceptor blockade on resting and hyperemic myocardial blood flow in normal humans. Myocardial blood flow, at baseline and after dipyridamole, was measured with positron emission tomography and 15O-labeled water in 11 normal volunteers at control and during alpha 1-blockade with doxazosin. Baseline myocardial blood flow during alpha 1-blockade was not different from control, whereas coronary resistance was significantly lower (73.48 +/- 18.31 vs. 89.84 +/- 27.96 mmHg.min.ml-1.g-1; P < 0.05). After dipyridamole, myocardial blood flow during alpha 1-blockade was significantly higher (3.50 +/- 0.75 vs. 2.58 +/- 0.54 ml.min-1.g-1; P < 0.01) and coronary resistance lower (25.30 +/- 7.37 vs. 33.89 +/- 7.04 mmHg.min.ml-1.g-1; P < 0.01) compared with control. In conclusion, in normal humans, dipyridamole-induced increase in myocardial blood flow is limited by alpha 1-mediated coronary vasoconstriction.

  7. Alpha-1 adrenergic receptors in the medial preoptic area are involved in the induction of sleep.

    PubMed

    Kumar, Velayudhan Mohan; Vetrivelan, Ramalingam; Mallick, Hruda Nanda

    2006-08-01

    This paper reviews the recent studies that led to the conclusion that the noradrenergic neurons projecting to the medial preoptic area (mPOA) are hypnogenic and that they mediate this action through alpha(1) adrenergic receptors. Microinjection of noradrenaline (NA) into the mPOA induced arousal. Studies using alpha(2) adrenergic drugs showed that the arousal induced by intrapreoptic injection of NA was due to its action on presynaptic alpha(2) adrenergic receptors. A combination of lesion and chemical stimulation techniques demonstrated that when NA acted on the postsynaptic alpha(1 )receptors in the mPOA, it induced sleep. Intrapreoptic injection of alpha(1) agonist, methoxamine could induce sleep, when the hypothermia, which was simultaneously produced, was behaviorally compensated for by the animal. Increased arousal produced by the destruction of noradrenergic fibers in the mPOA further confirmed the hypnogenic role of these fibers.

  8. Proteomic Identification of IPSE/alpha-1 as a Major Hepatotoxin Secreted by Schistosoma mansoni Eggs

    PubMed Central

    Abdulla, Maha-Hamadien; Lim, Kee-Chong; McKerrow, James H.; Caffrey, Conor R.

    2011-01-01

    Background Eggs deposited in the liver of the mammalian host by the blood fluke parasite, Schistosoma mansoni, normally drive a T-helper-2 (Th2)-mediated granulomatous response in immune-competent mice. By contrast, in mice deprived of T-cells and incapable of producing granulomata, egg-secreted proteins (ESP) induce acute hepatic injury and death. Previous work has shown that one such ESP, the T2 ribonuclease known as omega-1, is hepatotoxic in vivo in that specific antisera to omega-1 prevent hepatocyte damage. Methodology/Principal Findings Using an in vitro culture system employing mouse primary hepatocytes and alanine transaminase (ALT) activity as a marker of heptocyte injury, we demonstrated that S. mansoni eggs, egg-secreted proteins (ESP), soluble-egg antigen (SEA), and omega-1 are directly hepatotoxic and in a dose-dependent manner. Depletion of omega-1 using a monoclonal antibody abolished the toxicity of pure omega-1 and diminished the toxicity in ESP and SEA by 47 and 33%, respectively. Anion exchange chromatography of ESP yielded one predominant hepatotoxic fraction. Proteomics of that fraction identified the presence of IPSE/alpha-1 (IL-4 inducing principle from S. mansoni eggs), a known activator of basophils and inducer of Th2-type responses. Pure recombinant IPSE/alpha-1 also displayed a dose-dependent hepatotoxicity in vitro. Monoclonal antibody depletion of IPSE/alpha-1 abolished the latter's toxicity and diminished the total toxicity of ESP and SEA by 32 and 35%, respectively. Combined depletion of omega-1 and IPSE/alpha-1 diminished hepatotoxicity of ESP and SEA by 60 and 58% respectively. Conclusions We identified IPSE/alpha-1 as a novel hepatotoxin and conclude that both IPSE/alpha-1 and omega-1 account for the majority of the hepatotoxicity secreted by S. mansoni eggs. PMID:22039561

  9. Autoradiographic analysis of alpha 1-noradrenergic receptors in the human brain postmortem. Effect of suicide

    SciTech Connect

    Gross-Isseroff, R.; Dillon, K.A.; Fieldust, S.J.; Biegon, A. )

    1990-11-01

    In vitro quantitative autoradiography of alpha 1-noradrenergic receptors, using tritiated prazosin as a ligand, was performed on 24 human brains postmortem. Twelve brains were obtained from suicide victims and 12 from matched controls. We found significant lower binding to alpha 1 receptors in several brain regions of the suicide group as compared with matched controls. This decrease in receptor density was evident in portions of the prefrontal cortex, as well as the temporal cortex and in the caudate nucleus. Age, sex, presence of alcohol, and time of death to autopsy did not affect prazosin binding, in our sample, as measured by autoradiography.

  10. Evidence for an age-dependent functional expression of alpha 1D-adrenoceptors in the rat vasculature.

    PubMed

    Ibarra, M; Terrón, J A; López-Guerrero, J J; Villalobos-Molina, R

    1997-03-19

    The role of the alpha 1-adrenoceptor subtypes, and their possible change with maturation, in alpha 1-adrenoceptor-induced pressor responses in the rat has not been established. Thus, the effects of the alpha 1D-, alpha 1A/1D- and alpha 1B/1D-adrenoceptor antagonists, BMY 7378 (8-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl) 8-azaspiro (4.5) decane-7,9-dione 2HCl), 5-methyl-urapidil and chloroethylclonidine, respectively, on the pressor responses induced by phenylephrine in 1- and 5-month-old pithed rats were investigated. The pressor responses induced by phenylephrine were competitively antagonized by both BMY 7378 and chloroethylclonidine in 5-month-old, but not in young immature animals; in marked contrast, 5-methylurapidil antagonized with similar potency the phenylephrine-induced pressor responses in animals of both ages. The present pharmacological data suggest that functional expression of alpha 1D-adrenoceptors in the rat resistance vessels increases with age; alpha 1A-, but not alpha 1B- or alpha 1D-adrenoceptors, seem to predominate in immature animals. These findings represent the first evidence that age-related changes in functional alpha 1-adrenoceptor subtypes occur in the systemic vasculature in vivo. PMID:9098690

  11. alpha1-Microglobulin as a promising marker of cadmium-induced tubular dysfunction, possibly better than beta2-microglobulin.

    PubMed

    Moriguchi, J; Ezaki, T; Tsukahara, T; Furuki, K; Fukui, Y; Okamoto, S; Ukai, H; Sakurai, H; Ikeda, M

    2004-03-14

    The purpose of the present study was to evaluate the validity of alpha1-microglobulin (alpha1-MG) in comparison with popularly used beta2-microglobulin (beta2-MG). A database on 8975 cases of never-smoking adult women was revisited; the data were based on spot urine samples from the women in 10 prefectures all over Japan. The validity of alpha1-MG was examined following essentially the same protocol as beta2-MG was examined in a previous study. Comparisons were made for alpha1-MG as observed (e.g. alpha1-MG(ob)), as corrected for creatinine (CR or cr) (e.g. alpha1-MGcr) and as corrected for a specific gravity (SG or sg) of 1.016 (e.g. alpha1-MGsg). A cut-off value of 5.0 mg alpha1-MG/g cr or l was deduced from 400 microg beta2-MG/g cr taking advantage of the regression equation between alpha1-MG and beta2-MG. The prevalence of alph1-microglobulinuria as corrected for a specific gravity of 1.016 (or alpha1-MGsg-uria in short) was essentially unchanged irrespective of SG, except for in very dense or very thin urine samples. alpha1-MGcr-uria prevalence decreased at higher CR. Comparison of the present observation with previous findings on beta2-MG-uria prevalence showed that the variation in prevalence of MG-uria as a function of urine density was smaller for alpha1-MGsg whereas it was substantially larger for beta2-MGcr, and thus it appeared prudent to consider alpha1-MGsg rather than beta2-MGcr as a marker of tubular dysfunction.

  12. Protons inhibit Cl- conductance by direct or allosteric interaction with the GABA-binding site in the rat recombinant alpha1beta2gamma2L and alpha1beta2 GABAA receptor.

    PubMed

    Wang, Ming-De; Rahman, Mozibur; Zhu, Di

    2005-12-28

    Functional roles of external pH on the Cl- conductance were examined on Xenopus oocytes expressing rat recombinant alpha1beta2gamma2L and alpha1beta2 GABAA receptors. Acidic pH inhibited GABA-response in a reversible and concentration-dependent manner, significantly increasing the EC50 without appreciably changing the slope or maximal currents induced by GABA in the alpha1beta2gamma2L and alpha1beta2 receptors. In contrast, protonation did not influence the pentobarbital-gated currents in the alpha1beta2gamma2L receptors, suggesting that protons do not modulate channel activity by directly affecting the channel gating process. Protons competitively inhibited the bicuculline-induced antagonism on GABA in the alpha1beta2gamma2L receptors. The data support the hypothesis that protons inhibit GABAA receptor function by direct or allosteric interaction with the GABA-binding site.

  13. 21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... diabetes. (b) Classification. Class I (general controls). The device is exempt from the...

  14. 21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... diabetes. (b) Classification. Class I (general controls). The device is exempt from the...

  15. 21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... diabetes. (b) Classification. Class I (general controls). The device is exempt from the...

  16. Alpha 1-acid glycoprotein has immunomodulatory effects in neonatal swine adipose tissue

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alpha 1-acid glycoprotein (AGP) is the most abundant protein in serum of neonatal swine. This protein functions as an immunomodulator in the pig. Recent work has demonstrated that adipose tissue can express AGP mRNA, as well as numerous cytokine mRNA. The present study was designed to determine i...

  17. Regulation of alpha-1 acid glycoprotein synthesis by porcine hepatocytes in monolayer culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alpha 1-acid glycoprotein (AGP, ORM-1) is a highly glycosylated mammalian acute phase protein, which is synthesized primarily in the liver and represents the major serum protein in newborn pigs. Recent data have suggested that the pig is unique in that AGP is a negative acute phase protein in this ...

  18. Responsiveness of alpha 1 and beta 1 cochlear Na, K-ATPase isoforms to thyroid hormone.

    PubMed

    Zuo, J; Rarey, K E

    1996-05-01

    The effects of thyroid hormone on Na, K-ATPase subunit isoforms under euthyroid (EUTH), hypothyroid (HYPO) and hyperthyroid (HYPER) states were investigated via immunocytochemistry and the use of polyclonal antibodies specific to each isoform (alpha 1, alpha 2, alpha 3 and beta 1, beta 2). In HYPO animals, there was a distinct decrease in Na, K-ATPase alpha 1 isoform immunoreactivity in the stria vascularis (SV), spiral ganglion (SG) cells, spiral limbus (SLi) and cochlear nerve (CN) as compared with that in EUTH animals by the 17th day of the experiment. Immunostaining of the alpha 1 isoform increased in HYPER animals as compared with that in HYPO animals, and reached a level comparable to that in EUTH animals after 2 days of triiodothyronine (T3) treatment. Levels of alpha 2, alpha 3 and beta 2 isoforms did not appear to be affected by T3 administration. By the 19th day of a low I2 diet, the immunoreactive intensity of the beta 1 isoform was reduced in cochlear tissues of HYPO animals as compared with that in EUTH animals. The immunoreactivity of the beta 1 isoform increased after treatment with T3 for 4 days and was comparable with levels in EUTH animals. These data indicate that alpha 1 and beta 1 isoforms within specific cochlear regions of the adult rat are responsive to thyroid hormone.

  19. 21 CFR 866.5080 - Alpha-1-antichymotrypsin immunological test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alpha-1-antichymotrypsin immunological test system. 866.5080 Section 866.5080 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  20. 21 CFR 866.5080 - Alpha-1-antichymotrypsin immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alpha-1-antichymotrypsin immunological test system. 866.5080 Section 866.5080 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  1. 21 CFR 866.5080 - Alpha-1-antichymotrypsin immunological test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alpha-1-antichymotrypsin immunological test system. 866.5080 Section 866.5080 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  2. 21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  3. 21 CFR 866.5420 - Alpha-1-glycoproteins immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alpha-1-glycoproteins immunological test system. 866.5420 Section 866.5420 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  4. 21 CFR 866.5080 - Alpha-1-antichymotrypsin immunological test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alpha-1-antichymotrypsin immunological test system. 866.5080 Section 866.5080 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems §...

  5. The pharmacology of spontaneously open alpha 1 beta 3 epsilon GABA A receptor-ionophores.

    PubMed

    Maksay, Gábor; Thompson, Sally A; Wafford, Keith A

    2003-06-01

    Human alpha(1)beta(3) epsilon GABA(A) receptors were expressed in Xenopus oocytes and examined using the conventional two-electrode voltage-clamp technique and compared to alpha(1)beta(3)gamma(2) receptors. The effects of several GABA(A) agonists were studied, and the allosteric modulation of the channel by a number of GABAergic modulators investigated. The presence of the epsilon subunit increased the potency and efficacy of direct activation by partial GABA(A) agonists (piperidine-4-sulphonic acid and thio-4-PIOL), pentobarbital and neuro-steroids. Direct activation by 3-hydroxylated neurosteroids was restricted to 3alpha epimers, while chirality at C5 was indifferent. The 3beta-sulfate esters of pregnenolone and dehydroepiandrosterone inhibited the spontaneous currents with efficacies higher, while bicuculline methiodide and SR 95531 did so lower than picrotoxin and TBPS. Furosemide, fipronil, triphenylcyanoborate and Zn(2+) blocked the spontaneous currents of alpha(1)beta(3) epsilon receptors with different efficacies. Flunitrazepam and 4'-chlorodiazepam inhibited the spontaneous currents with micromolar potencies. In conclusion, spontaneously active alpha(1)beta(3) epsilon GABA(A) receptors can be potentiated and blocked by GABAergic agents within a broad range of efficacy.

  6. Mice lacking alpha 1 (IX) collagen develop noninflammatory degenerative joint disease.

    PubMed Central

    Fässler, R; Schnegelsberg, P N; Dausman, J; Shinya, T; Muragaki, Y; McCarthy, M T; Olsen, B R; Jaenisch, R

    1994-01-01

    Type IX collagen is a nonfibrillar collagen composed of three gene products, alpha 1(IX), alpha 2(IX), and alpha 3(IX). Type IX molecules are localized on the surface of type II-containing fibrils and consist of two arms, a long arm that is crosslinked to type II collagen and a short arm that projects into the perifibrillar space. In hyaline cartilage, the alpha 1(IX) collagen transcript encodes a polypeptide with a large N-terminal globular domain (NC4), whereas in many other tissues an alternative transcript encodes an alpha 1(IX) chain with a truncated NC4 domain. It has been proposed that type IX molecules are involved in the interaction of fibrils with each other or with other components of the extracellular matrix. To test this hypothesis, we have generated a mouse strain lacking both isoforms of the alpha 1(IX) chain. Homozygous mutant mice are viable and show no detectable abnormalities at birth but develop a severe degenerative joint disease resembling human osteoarthritis. Images PMID:8197187

  7. Role of alpha1-blockade in congenital long QT syndrome: investigation by exercise stress test.

    PubMed

    Furushima, H; Chinushi, M; Washizuka, T; Aizawa, Y

    2001-07-01

    Beta-blockade is widely reported to reduce the incidence of syncope in 75-80% of patients with congenital long QT syndrome (LQTS). However, despite full-dose beta-blockade, 20-25% of patients continue to have syncopal episodes and remain at high risk for sudden cardiac death. In some patients refractory to beta-blockade, the recurrence of arrhythmias is successfully prevented by left stellate ganglionectomy, and also by labetalol, a nonselective beta-blockade with alpha1-blocking action. These observations suggest that not only beta-adrenoceptors, but also alpha1-adrenoceptors, play an important pathogenic role, especially under sympathetic stimulation, in LQTS. The clinical effects of alpha1-blockade in congenital LQTS were investigated in 8 patients with familial or sporadic LQTS. Two measurements of the QT interval were taken, from the QRS onset to the T wave offset (QT) and from the QRS onset to the peak of the T wave (QTp). Using the Bruce protocol, an exercise test was performed after administration of beta-blockade alone and again after administration of alpha1-blockade. The following were compared: (1) Bazzet-corrected QT (QTc) and QTp (QTpc) intervals in the supine and standing position before exercise and in the early recovery phase after exercise; and (2) the slopes (reflecting the dynamic change in the QT interval during exercise) of the QT interval to heart rate were obtained from the linear regression during the exercise test. In the supine position before exercise, there was no change in the QTc before or after the addition of alpha1-blockade (498+/-23 vs 486+/-23 ms [NS]). However, in the upright position before exercise and in the early recovery phase after exercise, QTc was significantly shortened from 523+/-21 to 483+/-22ms (p<0.01), and from 521+/-30 to 490+/-39ms (p<0.01), respectively, by alpha1-blockade. The QTpc was unchanged in any situation. Consequently, QTc-QTpc was significantly shortened by alpha1-blockade in the upright position

  8. Possible dopaminergic stimulation of locus coeruleus alpha1-adrenoceptors involved in behavioral activation.

    PubMed

    Lin, Yan; Quartermain, David; Dunn, Adrian J; Weinshenker, David; Stone, Eric A

    2008-07-01

    alpha(1)-Adrenoceptors of the locus coeruleus (LC) have been implicated in behavioral activation in novel surroundings, but the endogenous agonist that activates these receptors has not been established. In addition to the canonical activation of alpha(1)-receptors by norepinephrine (NE), there is evidence that dopamine (DA) may also activate certain brain alpha(1)-receptors. This study examined the contribution of DA to exploratory activity in a novel cage by determining the effect of infusion of various dopaminergic and adrenergic drugs into the mouse LC. It was found that the D2/D3 agonist, quinpirole, which selectively blocks the release of CNS DA, produced a dose-dependent and virtually complete abolition of exploration and all movement in the novel cage test. The quinpirole-induced inactivity was significantly attenuated by coinfusion of DA but not by the D1 agonist, SKF38390. Furthermore, the DA attenuation of quinpirole inactivity was blocked by coinfusion of the alpha(1)-adrenergic receptor antagonist, terazosin, but not by the D1 receptor antagonist, SCH23390. LC infusions of either quinpirole or terazosin also produced profound inactivity in DA-beta-hydroxylase knockout (Dbh -/-) mice that lack NE, indicating that their behavioral effects were not due to an alteration of the release or action of LC NE. Measurement of endogenous DA, NE, and 5HT and their metabolites in the LC during exposure to the novel cage indicated an increase in the turnover of DA and NE but not 5HT. These results indicate that DA is a candidate as an endogenous agonist for behaviorally activating LC alpha(1)-receptors and may play a role in the activation of this nucleus by novel surroundings. PMID:18435418

  9. Cervical mucins carry alpha(1,2)fucosylated glycans that partly protect from experimental vaginal candidiasis.

    PubMed

    Domino, Steven E; Hurd, Elizabeth A; Thomsson, Kristina A; Karnak, David M; Holmén Larsson, Jessica M; Thomsson, Elisabeth; Bäckström, Malin; Hansson, Gunnar C

    2009-12-01

    Cervical mucins are glycosylated proteins that form a protective cervical mucus. To understand the role of mucin glycans in Candida albicans infection, oligosaccharides from mouse cervical mucins were analyzed by liquid chromatography-mass spectrometry. Cervical mucins carry multiple alpha(1-2)fucosylated glycans, but alpha(1,2)fucosyltransferase Fut2-null mice are devoid of these epitopes. Epithelial cells in vaginal lavages from Fut2-null mice lacked Ulex europaeus agglutinin-1 (UEA-I) staining for alpha(1-2)fucosylated glycans. Hysterectomy to remove cervical mucus eliminated UEA-I and acid mucin staining in vaginal epithelial cells from wild type mice indicating the cervix as the source of UEA-I positive epithelial cells. To assess binding of alpha(1-2) fucosylated glycans on C. albicans infection, an in vitro adhesion assay was performed with vaginal epithelial cells from wild type and Fut2-null mice. Vaginal epithelial cells from Fut2-null mice were found to bind increased numbers of C. albicans compared to vaginal epithelial cells obtained from wild type mice. Hysterectomy lessened the difference between Fut2-null and wild type mice in binding of C. ablicans in vitro and susceptibility to experimental C. albicans vaginitis in vivo. We generated a recombinant fucosylated MUC1 glycanpolymer to test whether the relative protection of wild type mice compared to Fut2-null mice could be mimicked with exogenous mucin. While a small portion of the recombinant MUC1 epitopes displayed alpha(1-2)fucosylated glycans, the predominant epitopes were sialylated due to endogenous sialyltransferases in the cultured cells. Intravaginal instillation of recombinant MUC1 glycanpolymer partially reduced experimental yeast vaginitis suggesting that a large glycanpolymer, with different glycan epitopes, may affect fungal burden.

  10. Quantitation of residual trypsin in cell-based therapeutics using immobilized α-1-antitrypsin or SBTI in an ELISA format.

    PubMed

    Braatz, James A; Elias, Christopher; Finny, Joseph G; Tran, Huan; McCaman, Michael

    2015-02-01

    An Enzyme-Linked Immunosorbent Assay (ELISA) has been developed for the quantitation of porcine trypsin as a process residual in cell therapy products based on its capture by either of two immobilized anti-trypsins, α-1-antitrypsin (α1AT) or soybean trypsin inhibitor (SBTI) followed by detection with a polyclonal goat anti-porcine trypsin-IgG conjugated with peroxidase. It was demonstrated that an extended range of antigen quantitation could be achieved that covered nearly three orders of magnitude of trypsin concentration. The utility of the assay was demonstrated by its application to samples generated in a cell-based therapeutic manufacturing setting.

  11. The lipocalin alpha1-microglobulin protects erythroid K562 cells against oxidative damage induced by heme and reactive oxygen species.

    PubMed

    Olsson, Magnus G; Olofsson, Tor; Tapper, Hans; Akerstrom, Bo

    2008-08-01

    Alpha(1)-microglobulin is a 26 kDa plasma and tissue glycoprotein that belongs to the lipocalin protein superfamily. Recent reports show that it is a reductase and radical scavenger and that it binds heme and has heme-degrading properties. This study has investigated the protective effects of alpha(1)-microglobulin against oxidation by heme and reactive oxygen species in the human erythroid cell line, K562. The results show that alpha(1)-microglobulin prevents intracellular oxidation and up-regulation of heme oxygenase-1 induced by heme, hydrogen peroxide and Fenton reaction-generated hydroxyl radicals in the culture medium. It also reduces the cytosol of non-oxidized cells. Endogeneous expression of alpha(1)-microglobulin was up-regulated by these oxidants and silencing of the alpha(1)-microglobulin expression increased the cytosol oxidation. alpha(1)-microglobulin also inhibited cell death caused by heme and cleared cells from bound heme. Binding of heme to alpha(1)-microglobulin increased the radical reductase activity of the protein as compared to the apo-protein. Finally, alpha(1)-microglobulin was localized mainly at the cell surface both when administered exogeneously and in non-treated cells. The results suggest that alpha(1)-microglobulin is involved in the defence against oxidative cellular injury caused by haemoglobin and heme and that the protein may employ both heme-scavenging and one-electron reduction of radicals to achieve this.

  12. Alpha-1-adrenergic receptors in heart failure: the adaptive arm of the cardiac response to chronic catecholamine stimulation.

    PubMed

    Jensen, Brian C; OʼConnell, Timothy D; Simpson, Paul C

    2014-04-01

    Alpha-1-adrenergic receptors (ARs) are G protein-coupled receptors activated by catecholamines. The alpha-1A and alpha-1B subtypes are expressed in mouse and human myocardium, whereas the alpha-1D protein is found only in coronary arteries. There are far fewer alpha-1-ARs than beta-ARs in the nonfailing heart, but their abundance is maintained or increased in the setting of heart failure, which is characterized by pronounced chronic elevation of catecholamines and beta-AR dysfunction. Decades of evidence from gain and loss-of-function studies in isolated cardiac myocytes and numerous animal models demonstrate important adaptive functions for cardiac alpha-1-ARs to include physiological hypertrophy, positive inotropy, ischemic preconditioning, and protection from cell death. Clinical trial data indicate that blocking alpha-1-ARs is associated with incident heart failure in patients with hypertension. Collectively, these findings suggest that alpha-1-AR activation might mitigate the well-recognized toxic effects of beta-ARs in the hyperadrenergic setting of chronic heart failure. Thus, exogenous cardioselective activation of alpha-1-ARs might represent a novel and viable approach to the treatment of heart failure.

  13. Biosynthesis and regulation of rat alpha 1-inhibitor3, a negative acute-phase reactant of the macroglobulin family.

    PubMed Central

    Geiger, T; Lamri, Y; Tran-Thi, T A; Gauthier, F; Feldmann, G; Decker, K; Heinrich, P C

    1987-01-01

    The biosynthesis of rat alpha 1-inhibitor3, a negative acute-phase reactant specifically found in rodents, was studied in vitro in a cell-free translation system from rabbit reticulocytes, in rat hepatocyte primary cultures and in vivo by immunocytochemistry using normal and turpentine-injected rats. By sucrose-gradient centrifugation and subsequent translation of the fractionated RNA in vitro it was found that the mRNA coding for alpha 1-inhibitor3 exhibited a size of about 28S. For the alpha 1-inhibitor3 translated in vitro an apparent Mr of 155,000 was determined. A continuous decrease in the level of alpha 1-inhibitor3 in serum during experimental inflammation induced by turpentine injection was demonstrated by means of quantitative 'rocket' immunoelectrophoresis. This result agrees with the observation by immunocytochemistry of a drastic decrease in alpha 1-inhibitor3 levels in hepatocytes 24 h after turpentine injection. At that time alpha 1-inhibitor3 is mainly located in the Golgi apparatus, whereas it is also present in the membranes of the rough and smooth endoplasmic reticulum when normal liver is used. All hepatocytes, but no other hepatic cells, contain alpha 1-inhibitor3. When hepatocyte primary cultures were labelled with [35S]methionine and alpha 1-inhibitor3 was immunoprecipitated from the hepatocyte medium and the supernatant of homogenized cells, two different forms of alpha 1-inhibitor3 were found. The intracellular form of alpha 1-inhibitor3, with an apparent Mr of 173,000, is characterized by oligosaccharide side chains of the high-mannose type. The form of alpha 1-inhibitor3 in the medium exhibited an Mr of 186,000 and carried carbohydrate side chains of the complex type. After labelling hepatocytes with radioactive sugars, [3H]mannose was found in both forms of alpha 1-inhibitor3, whereas [3H]fucose and [3H]galactose were incorporated only into the form found in the medium. In the presence of tunicamycin an unglycosylated alpha 1-inhibitor3

  14. Different effects of the glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine on the glycosylation of rat alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein.

    PubMed

    Gross, V; Tran-Thi, T A; Schwarz, R T; Elbein, A D; Decker, K; Heinrich, P C

    1986-06-15

    The glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine were used to inhibit oligosaccharide processing in primary cultures of rat hepatocytes. Their effect on the glycosylation of alpha 1-proteinase inhibitor (alpha 1PI) and alpha 1-acid glycoprotein (alpha 1AGP) was studied. Of the three glucosidase inhibitors examined, 1-deoxynojirimycin inhibited not only oligosaccharide trimming but also glycosylation de novo of newly synthesized proteins, resulting in the formation of alpha 1PI with two and three (normally carrying three) and alpha 1AGP with two to five (normally carrying six) oligosaccharide side chains. In the presence of the glucosidase inhibitors, glucosylated high-mannose-type oligosaccharides accumulated. Whereas most of the endoglucosaminidase-H-sensitive oligosaccharides formed in the presence of 1-deoxynojirimycin contained only one glucose residue, N-methyl-1-deoxynojirimycin and castanospermine led mainly to the formation of oligosaccharides with three glucose residues. None of the three glucosidase inhibitors completely prevented the formation of complex-type oligosaccharides. Thus, in their presence, alpha 1PI and alpha 1AGP with a mixture of both high-mannose and complex-type oligosaccharides were secreted. PMID:2947571

  15. Different effects of the glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine on the glycosylation of rat alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein.

    PubMed Central

    Gross, V; Tran-Thi, T A; Schwarz, R T; Elbein, A D; Decker, K; Heinrich, P C

    1986-01-01

    The glucosidase inhibitors 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin and castanospermine were used to inhibit oligosaccharide processing in primary cultures of rat hepatocytes. Their effect on the glycosylation of alpha 1-proteinase inhibitor (alpha 1PI) and alpha 1-acid glycoprotein (alpha 1AGP) was studied. Of the three glucosidase inhibitors examined, 1-deoxynojirimycin inhibited not only oligosaccharide trimming but also glycosylation de novo of newly synthesized proteins, resulting in the formation of alpha 1PI with two and three (normally carrying three) and alpha 1AGP with two to five (normally carrying six) oligosaccharide side chains. In the presence of the glucosidase inhibitors, glucosylated high-mannose-type oligosaccharides accumulated. Whereas most of the endoglucosaminidase-H-sensitive oligosaccharides formed in the presence of 1-deoxynojirimycin contained only one glucose residue, N-methyl-1-deoxynojirimycin and castanospermine led mainly to the formation of oligosaccharides with three glucose residues. None of the three glucosidase inhibitors completely prevented the formation of complex-type oligosaccharides. Thus, in their presence, alpha 1PI and alpha 1AGP with a mixture of both high-mannose and complex-type oligosaccharides were secreted. Images Fig. 1. Fig. 2. Fig. 5. PMID:2947571

  16. Enhancement of ovalbumin-induced pulmonary eosinophilia by intranasal administration of alpha1-proteinase inhibitor type 2 antisense oligonucleotides.

    PubMed

    Sung, Ha-Na; Jeon, Chang-Hwan; Gill, Byoung-Chul; Kim, Hye-Rin; Cheong, Sun-Woo; Park, Joo-Hung

    2009-01-29

    To identify asthma-susceptibility genes, we did proteome analyses of the lung from control and ovalbumin-sensitized BALB/c mice. Among the 6 up-regulated proteins is alpha(1)-protease inhibitor (alpha(1)-PI) type 2, which is a member of the serine protease inhibitor superfamily of protease inhibitors that participate in a variety of physiological functions, including extracellular matrix remodeling and inflammation. The up-regulated expression of alpha(1)-PI type 2 was confirmed by real-time PCR. Then we examined mRNA expression of five members of the alpha(1)-PI family genes (alpha(1)-PI types 1-5) in several organs of BALB/c mice and found that in addition to the liver, all the organs tested also expressed different isoforms of alpha(1)-PI in a tissue-specific manner, albeit to a lesser extent compared with the liver. When a similar study was performed with C57BL/6 mice, which have been shown to be more susceptible to ovalbumin-induced asthma than BALB/c mice, a pair of remarkable differences between the mouse strains were revealed: (1) the magnitude of alpha(1)-PI type 2 mRNA in all the organs was much higher in BALB/c than in C57BL/6 mice and (2) alpha(1)-PI type 2 is the only isoform expressed in the lung of BALB/c, but not of C57BL/c mice. Using the antisense oligonucleotide technology to specifically down-regulate expression of alpha(1)-PI type 2, we demonstrated that pulmonary infiltration of eosinophils was significantly increased by intranasal administration of alpha(1)-PI type 2 antisense oligonucleotides in OVA-sensitized mice, suggesting that alpha(1)-PI type 2 may suppress the progress of asthma, probably by acting on neutrophil elastase, which can produce many of the pathological features of asthma.

  17. Up-regulation of alpha1-microglobulin by hemoglobin and reactive oxygen species in hepatoma and blood cell lines.

    PubMed

    Olsson, Magnus G; Allhorn, Maria; Olofsson, Tor; Akerström, Bo

    2007-03-15

    alpha(1)-Microglobulin is a 26-kDa glycoprotein synthesized in the liver, secreted to the blood, and rapidly distributed to the extravascular compartment of all tissues. Recent results show that alpha(1)-microglobulin has heme-binding and heme-degrading properties and it has been suggested that the protein is involved in the defense against oxidation by heme and reactive oxygen species. In the present study the influence of hemoglobin and reactive oxygen species (ROS) on the cellular expression of alpha(1)-microglobulin was investigated. Oxy- and methemoglobin, free heme, and Fenton reaction-induced hydroxyl radicals induced a dose-dependent up-regulation of alpha(1)-microglobulin on both mRNA and protein levels in hepatoma cells and an increased secretion of alpha(1)-microglobulin. The up-regulation was reversed by the addition of catalase and ascorbate, and by reacting hemoglobin with cyanide which prevents redox reactions. Furthermore, the blood cell lines U937 and K562 expressed alpha(1)-microglobulin at low levels, and this expression increased up to 11-fold by the addition of hemoglobin. These results suggest that alpha(1)-microglobulin expression is induced by ROS, arising from redox reactions of hemoglobin or from other sources and are consistent with the hypothesis that alpha(1)-microglobulin participates in the defense against oxidation by hemoglobin, heme, and reactive oxygen species.

  18. Altered hepatic vasopressin and alpha 1-adrenergic receptors after chronic endotoxin infusion

    SciTech Connect

    Roth, B.L.; Spitzer, J.A.

    1987-05-01

    Sepsis and septic shock are complicated by a number of hemodynamic and metabolic aberrations. These include catecholamine refractoriness and altered glucose metabolism. Recently, a nonshock rat model of continuous endotoxin infusion via an implanted osmotic pump was developed that reproduces some of the metabolic and cardiovascular findings of human sepsis. By using this model, we have found a decreased number of hepatic plasma membrane alpha 1-adrenergic and (Arg8)vasopressin receptors in rats continuously infused with endotoxin. There was a significant decrease in (/sup 3/H)prazosin (35 +/- 7%) and (/sup 3/H) (Arg8)vasopressin (43 +/- 8%) receptors after 30 h of continuous endotoxin infusion with no change in affinity. The ability of norepinephrine to form the high-affinity complex with alpha 1-adrenergic receptors was not altered after chronic endotoxin infusion. The results are consistent with the concept that alterations in receptor number might underlie certain of the metabolic consequences of chronic sepsis.

  19. Synergistic alpha-1 and alpha-2 adrenergic stimulation of rat proximal nephron Na+/H+ exchange

    SciTech Connect

    Gesek, F.A.; Cragoe, E.J. Jr.; Strandhoy, J.W.

    1989-06-01

    Both alpha-1 and alpha-2 adrenoceptors have been localized to the renal cortex, with the majority of binding sites on the proximal tubule. Because the major regulator of Na+ uptake into the proximal tubule is the Na+/H+ exchanger, and because alpha-1 and alpha-2 adrenoceptors stimulate it in other tissues, we tested the hypothesis that both alpha adrenoceptor subtypes can increase Na+ uptake into the proximal nephron by stimulating the Na+/H+ antiporter. Enhancement of Na+ transport by agonists was studied in isolated rat proximal tubules by determining the uptake of 22Na that was suppressible by the Na+/H+ inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA). The phorbol ester, phorbol-12-myristate-13-acetate, (0.1 microM), directly stimulated the antiporter through protein kinase C and increased EIPA-suppressible 22Na uptake 250% above control. The alpha-1 adrenoceptor agonists, cirazoline and phenylephrine, in addition to the mixed agonist, norepinephrine, maximally stimulated uptake by 226 to 232% at 1 microM concentrations. alpha-2 agonists produced a range of maximal stimulations at 1 microM from 65% with guanabenz to 251% with B-HT 933. Increases in 22Na uptake by agonists were inhibited by selective adrenergic antagonists and by EIPA. The drugs did not change the EIPA-resistant component of 22Na uptake. Inasmuch as the adrenoceptor subtypes likely stimulated Na+/H+ exchange by differing intracellular pathways impinging upon common transport steps, we examined whether simultaneous stimulation of both pathways was additive. Submaximal concentrations (5 nM each) of alpha-1 and alpha-2 adrenoceptor agonists in combination synergistically enhanced 22Na uptake to a level similar to 1 microM concentrations of adrenoceptor agonists alone or in combination.

  20. Relationship between alpha-1 receptors and cations in rat liver plasma membranes

    SciTech Connect

    Smart, J.L.

    1986-01-01

    The influence of cations on binding of (/sup 3/H)-prazosin (PRZ), an alpha-1 specific antagonist, to alpha receptor sites in rat liver plasma membranes was examined. All cations tested were able to produce dose-dependent shifts to lower affinity binding sites for PRZ. The maximum number of binding sites was also observed to be altered. Inclusion of cations resulted in a slower observed rate constant for association as well as a delay in the dissociation of specifically bound PRZ following the addition of phentolamine. In contrast, the ability of (-)-norepinephrine to displace PRZ was enhanced by the addition of cations. The influence of alpha-1 receptor stimulation on Na/sup +//K/sup +/-ATPase activity in rat liver was examined by two methods - rat liver plasma membrane Na/sup +//K/sup +/-ATPase activity following liver perfusion in situ and /sup 86/Tb uptake in rat liver slices. The activity of the Na/sup +/ pump was found to be biphasic following exposure to phenylephrine (PE), an alpha-1 agonist. Stimulation (35%) was present over the first two minutes, while activity was inhibited over the interval of 5 to 10 minutes of continued PE exposure. Both phases were blocked by prazosin. The influence of DAG and protein kinase C (PKC) in alpha-1 receptor modulation of the Na/sup +/ pump was studied by employing 4-beta-phorbol (PMA), a phorbol ester which activates PKC. Perfusion of livers with PMA in situ or incubation with slices yielded inhibition of ATPase activity in membranes and /sup 86/Rb uptake in that was qualitatively and quantitatively similar to PE. These results suggest cations may influence receptor function in vivo and in vitro and the inhibitory effects of PE on the sodium pump may be mediated through PKC.

  1. Association of endodermal sinus tumour, testicular teratoma and alpha1-fetoprotein.

    PubMed

    Gariépy, G; Lafortune, M; Poisson, R

    1976-08-01

    An endodermal sinus tumour in the retroperitoneal region was associated with the presence of alpha1-fetoprotein (AFP) in the patient's serum. At autopsy a simple cystic teratoma of the right testicle was also found. The association of these two tumours has been reported before. The classification of these malignant germ-cell tumours and an understanding of their evolution may be aided by the discovery that AFP is often found in the patient's serum.

  2. An increased expression of Ca(2+) channel alpha(1A) subunit immunoreactivity in deep cerebellar neurons of rolling mouse Nagoya.

    PubMed

    Sawada, K; Sakata-Haga, H; Ando, M; Takeda, N; Fukui, Y

    2001-12-01

    Rolling mouse Nagoya (RMN) is an ataxic mutant and carries a mutation in the gene coding for the alpha(1A) subunit of the P/Q-type Ca(2+) channel. We examined the immunohistochemical expression of the alpha(1A) subunit in deep cerebellar nuclei of RMN. The antibody used recognized residues 865-883 of the mouse alpha(1A) subunit not overlapping the altered sequences in RMN. In RMN, many neurons exhibited definite alpha(1A) subunit-staining in the medial nucleus, interposed nucleus, and lateral nucleus of deep cerebellar nuclei. The number of positive neurons in these nuclei was significantly higher in RMN than in controls. Increased expression of the alpha(1A) subunit in deep cerebellar neurons might compensate for the altered function of the P/Q-type Ca(2+) channel of RMN.

  3. Issues in pharmaceutical development of thymosin alpha1 from preclinical studies through marketing.

    PubMed

    Tuthill, Cynthia

    2007-09-01

    SciClone Pharmaceuticals licensed the commercial and patent rights to thymosin alpha1, for geographical regions of the world excluding the United States and Europe, in the early 1990s. With this license, SciClone embarked on global drug development, and the issues encountered for thymosin alpha1 are reflective of the roller coaster of modern approval of pharmaceuticals. Most of the required toxicology studies had been completed prior to licensure, but some newer studies had to be conducted to obtain approvals in certain countries. The recent development of the "International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use" (ICH) guidelines allows for a clearer definition of the required battery of toxicology studies, although some countries still have not adopted these guidelines, and the local regulations have had to be understood and followed. Other hurdles include the complications that manufacturing requirements can differ between countries, and certain countries require local clinical experience trials in addition to SciClone's cumulative clinical data. A further obstacle was the pleiotropic nature of the mechanism of action of thymosin alpha1, with the resulting difficulty in the unraveling of its pharmacologic effects. With close attention to these regulatory details, SciClone has obtained approvals in more than 30 countries and has successfully begun commercial sales.

  4. Issues in pharmaceutical development of thymosin alpha1 from preclinical studies through marketing.

    PubMed

    Tuthill, Cynthia

    2007-09-01

    SciClone Pharmaceuticals licensed the commercial and patent rights to thymosin alpha1, for geographical regions of the world excluding the United States and Europe, in the early 1990s. With this license, SciClone embarked on global drug development, and the issues encountered for thymosin alpha1 are reflective of the roller coaster of modern approval of pharmaceuticals. Most of the required toxicology studies had been completed prior to licensure, but some newer studies had to be conducted to obtain approvals in certain countries. The recent development of the "International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use" (ICH) guidelines allows for a clearer definition of the required battery of toxicology studies, although some countries still have not adopted these guidelines, and the local regulations have had to be understood and followed. Other hurdles include the complications that manufacturing requirements can differ between countries, and certain countries require local clinical experience trials in addition to SciClone's cumulative clinical data. A further obstacle was the pleiotropic nature of the mechanism of action of thymosin alpha1, with the resulting difficulty in the unraveling of its pharmacologic effects. With close attention to these regulatory details, SciClone has obtained approvals in more than 30 countries and has successfully begun commercial sales. PMID:17947591

  5. alpha-1 Adrenergic receptors stimulation induces the proliferation of neural progenitor cells in vitro.

    PubMed

    Hiramoto, Takeshi; Ihara, Yoshiaki; Watanabe, Yasuhiro

    2006-11-01

    The proliferation of neural progenitor cells (NPCs) is regulated by classical neurotransmitters such as dopamine, serotonin and acetylcholine, via its own receptors. Previous studies have reported that the depletion of L-norepinephrine decreases the proliferation of NPCs in the adult rat hippocampus and it has been suggested that L-norepinephrine regulates the proliferation of NPCs. However, it remains unknown whether or not adrenergic receptors are involved in the increased proliferation of NPCs. In the present study, an MTT cell proliferation assay was carried out in order to investigate the roles played by adrenergic receptors in the proliferation of NPCs. We demonstrated that L-epinephrine enhanced the proliferation of embryonic NPCs in vitro. In addition, the alpha-1 adrenergic receptor agonist L-phenylephrine was found to enhance the proliferation of NPCs, whereas an alpha-adrenergic antagonist and selective alpha-1 antagonists significantly inhibited cell proliferation increases induced by L-epinephrine and L-phenylephrine. These results suggest that stimulation with alpha-1 adrenergic receptors induces the proliferation of embryonic NPCs.

  6. Long-term reduction of intimal hyperplasia by the selective alpha-1 adrenergic antagonist doxazosin.

    PubMed

    Vashisht, R; Sian, M; Franks, P J; O'Malley, M K

    1992-12-01

    Studies have shown that alpha 1-adrenergic blockade reduces intimal hyperplasia in the rabbit aorta. In this study a selective alpha 1-adrenergic antagonist, doxazosin, has been used to examine whether this effect is persistent and dose dependent. Forty-eight New Zealand White rabbits underwent endothelial denudation of the abdominal aorta using a Fogarty balloon catheter. Test rabbits were given low-dose (2 mg) or high-dose (8 mg) doxazosin daily and all animals killed at either 1 or 12 weeks after the procedure. The aortas were harvested after fixation in situ with 4 per cent glutaraldehyde and neointimal hyperplasia was measured, using an x-y digitizer, as the percentage reduction in luminal cross-sectional area. At 1 week after surgery, rabbits receiving the low dose had a median area reduction of 7.7 per cent and those receiving the high dose a reduction of 8.2 per cent; both had significantly less intimal hyperplasia than control rabbits, which had a median area reduction of 14.8 per cent (P < 0.01). However, at 12 weeks, when compared with the 32.6 per cent reduction in the control group, only those rabbits receiving high-dose doxazosin had significantly less intimal hyperplasia, with a reduction of 5.5 per cent (P < 0.001). It is concluded that selective alpha 1-adrenergic blockade significantly reduces neointimal hyperplasia, that this effect is dose dependent, and that it persists for at least 3 months.

  7. Alternative nonallelic deletion is constitutive of ruminant alpha(s1)-casein.

    PubMed

    Ferranti, P; Lilla, S; Chianese, L; Addeo, F

    1999-07-01

    Multiple forms of alpha(s1)-casein were identified in the four major ruminant species by structural characterization of the protein fraction. While alpha(s1)-casein phenotypes were constituted by a mixture of at least seven molecular forms in ovine and caprine species, there were only two forms in bovine and water buffalo species. In ovine and caprine forms the main component corresponded to the 199-residue-long form, and the deleted proteins differed from the complete one by the absence of peptides 141-148, 110-117, or Gln78, or a combination of such deletions. The deleted segments corresponded to the sequence regions encoded by exons 13 and 16, and by the first triplet of exon 11 (CAG), suggesting that the occurrence of the short protein forms is due to alternative skipping, as previously demonstrated for some caprine and ovine phenotypes. The alternative deletion of Gln78 in alpha(s1)-casein, the only form common to the milk of all the species examined and located in a sequence region joining the polar phosphorylation cluster and the hydrophobic C-terminal domain of the protein, may play a functional role in the stabilization of the milk micelle structure.

  8. Analysis of the activity of alpha 1-adrenoceptor antagonists in rat aorta.

    PubMed Central

    Van der Graaf, P. H.; Shankley, N. P.; Black, J. W.

    1996-01-01

    1. In this study, the effect of seven alpha 1-adrenoceptor antagonists (tamsulosin, phentolamine, prazosin, WB-4101, 5-methylurapidil, spiperone and HV723) have been examined on the contractile response to noradrenaline (NA) and phenylephrine (PE) in rat isolated aorta. 2. NA and PE, when administered using a cumulative dosing schedule, both produced concentration-dependent contraction of aortic rings. It was possible to fit the individual concentration-effect (E/[A]) curve data to the Hill equation to provide estimates of the curve midpoint location (p[A]50 = 7.74 +/- 0.10 and 7.14 +/- 0.18), midpoint slope (nH = 0.82 +/- 0.03 and 0.99 +/- 0.10) and upper asymptote (alpha = 3.2 +/- 0.3 and 3.1 +/- 0.2 g) parameters for NA and PE, respectively. However, the Hill equation provided a better fit to the E/[A] curve data obtained with another contractile agent, 5-hydroxytryptamine (5-HT) (p[A50] = 6.09 +/- 0.08, nH = 1.49 +/- 0.09, alpha = 2.6 +/- 0.3 g), as judged by calculation of the mean sum of squares of the differences between the observed and predicted values. 3. All of the antagonists investigated produced concentration-dependent inhibition of the contractile responses of the aorta to NA and PE. Although no significant effects on the upper asymptotes of the E/[A] curves of any of the antagonists tested were detected, only tamsulosin and 5-methylurapidil did not have a significant effect on the slope (nH) of the NA and PE E/[A] curves. The other antagonists produced significant steepening of the curves obtained with NA and/or PE. 4. Notwithstanding the fact that one of the basic criteria for simple competitive antagonism at a single receptor class was not always satisfied, the individual log [A]50 values estimated in the absence and presence of antagonist within each experiment were fitted to the competitive model. The Schild plot slope parameters for the antagonism of NA and PE by phentolamine and HV723 were found to be significantly less than unity. The Schild

  9. Alpha1 and Alpha2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

    SciTech Connect

    Lochter, Andre; Navre, Marc; Werb, Zena; Bissell, Mina J

    1998-06-29

    Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits {alpha}6 and {beta}1, but not against {alpha}1 and {alpha}2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against {beta}1, but not against a6 or {alpha}2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against {alpha}1 integrins impaired only cell adhesion to type IV collagen. Antibodies against {alpha}1, {alpha}2, {alpha}6, and {beta}1, but not {alpha}5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins {alpha}1 and {alpha}2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against {alpha}1 and {alpha}2, but not {alpha}6 and {beta}1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against {alpha}1 and {alpha}2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-{alpha}6 antibodies. Our data indicate that {alpha}1 and {alpha}2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas {alpha}6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

  10. 1-deoxynojirimycin impairs oligosaccharide processing of alpha 1-proteinase inhibitor and inhibits its secretion in primary cultures of rat hepatocytes.

    PubMed

    Gross, V; Andus, T; Tran-Thi, T A; Schwarz, R T; Decker, K; Heinrich, P C

    1983-10-25

    1-Deoxynojirimycin was found to inhibit oligosaccharide processing of rat alpha 1-proteinase inhibitor. In normal hepatocytes alpha 1-proteinase inhibitor was present in the cells as a 49,000 Mr high mannose type glycoprotein with oligosaccharide side chains having the composition Man9GlcNAc and Man8GlcNAc with the former in a higher proportion. Hepatocytes treated with 5 mM 1-deoxynojirimycin accumulated alpha 1-proteinase inhibitor as a 51,000 Mr glycoprotein with carbohydrate side chains of the high mannose type, containing glucose as measured by their sensitivity against alpha-glucosidase, the largest species being Glc3Man9GlcNAc. Conversion to complex oligosaccharides was inhibited by the drug. In addition, increasing concentrations of 1-deoxynojirimycin inhibited glycosylation resulting in the formation of some alpha 1-proteinase inhibitor with two instead of three oligosaccharide side chains. 5 mM 1-deoxynojirimycin inhibited the secretion of alpha 1-proteinase inhibitor by about 50%, whereas secretion of albumin was unaffected. The oligosaccharides of alpha 1-proteinase inhibitor secreted from 1-deoxynojirimycin-treated cells were characterized by their susceptibility to endoglucosaminidase H, incorporation of [3H]galactose, and [3H]fucose and concanavalin A-Sepharose chromatography. It was found that 1-deoxynojirimycin did not completely block oligosaccharide processing, resulting in the formation of alpha 1-proteinase inhibitor molecules carrying one or two complex type oligosaccharides. Only these alpha 1-proteinase inhibitor molecules processed to the complex type in one or two of their oligosaccharide chains were nearly exclusively secreted. This finding demonstrates the importance of oligosaccharide processing for the secretion of alpha 1-proteinase inhibitor. PMID:6226656

  11. Effect of alpha1-adrenoceptor blockade on coronary vasodilator reserve in cardiac syndrome X.

    PubMed

    Rosen, S D; Lorenzoni, R; Kaski, J C; Foale, R A; Camici, P G

    1999-10-01

    We sought to test the response of the coronary microcirculation to alpha-adrenoceptor blockade in patients with syndrome X (angina, ischemia-like stress electrocardiogram, and a normal coronary arteriogram). The response of the microcirculation was assessed by quantification of coronary vasodilator reserve (the ratio of hyperemic to resting myocardial blood flow). We investigated 28 patients with syndrome X [18 women, age 54.4 (7.6) years]. Myocardial blood flow was measured at rest and after dipyridamole by using positron emission tomography with H(2)15O. The measurements were made before and after treatment for 10 days with doxazosin (1 mg o.d. for 3 days, followed by 2 mg o.d. for 7 days) or a matched placebo, similarly administered. Patients were randomized to alpha1-blockade or to placebo in double-blind fashion. No significant differences were demonstrable between syndrome X patients treated with doxazosin and those receiving placebo, with respect to resting myocardial blood flow, myocardial blood flow after dipyridamole, or coronary vasodilator reserve (the ratio of the latter two). In addition, no relations were demonstrable among myocardial blood flow, coronary vasodilator reserve, development of chest pain after dipyridamole, or development of ischemia-like ECG changes. Doxazosin had no effect on the perception of chest pain after dipyridamole. No differences were found between the effects of alpha1-blockade with doxazosin or those of placebo with respect to myocardial blood flow in syndrome X. The values obtained for myocardial blood flow and coronary vasodilator reserve for the patients were within the normal range. The data do not support the case for alpha1-mediated vasoconstriction having an etiologic role in the chest pain of syndrome X.

  12. Binding properties of alpha-1 adrenergic receptors in rat cerebral cortex: similarity to smooth muscle

    SciTech Connect

    Minneman, K.P.

    1983-12-01

    The characteristics of alpha-1 adrenergic receptors in rat cerebral cortex were examined using the radioiodinated alpha-1 adrenergic receptor antagonist ((/sup 125/I)BE). (/sup 125/I)BE labeled a single class of high-affinity binding sites in a particulate fraction of rat cerebral cortex with mass action kinetics and a KD of 57 pM. The binding of (/sup 125/I)BE was inhibited by various alpha adrenergic receptor antagonists, partial agonists and full agonists. The potency of these compounds in competing for the (/sup 125/I)BE binding sites suggested that (/sup 125/I)BE was labeling alpha-1 adrenergic receptors in rat cerebral cortex. In the absence of a physiological concentration of NaCl in the assay medium there was a small (20%) decrease in the density of (/sup 125/I)BE binding sites with no effect on the KD value. The absence of NaCl also caused a 4-fold increase in the potency of norepinephrine in competing for (/sup 125/I)BE binding sites. All drugs competed for (/sup 125/I) BE binding sites with Hill coefficients greater than 0.86, except for oxymetazoline which had a Hill coefficient of 0.77. Scatchard analysis of specific (/sup 125/I)BE binding in the presence of various competing drugs showed that the inhibition by both agonists and antagonists was purely competitive, but the inhibition by oxymetazoline was complex. Treatment of the particulate fraction of rat cerebral cortex with 0.2 to 200 nM phenoxybenzamine for 10 min caused a dose-dependent decrease in the density of (/sup 125/I) BE binding sites which could be mostly blocked by the presence of norepinephrine during the phenoxybenzamine exposure.

  13. Conformation Analysis of Peptides Derived from Laminin Alpha 1-2 Chain Using Molecular Dynamics Simulation

    NASA Astrophysics Data System (ADS)

    Yamada, Hironao; Fukuda, Masaki; Miyakawa, Takeshi; Morikawa, Ryota; Takasu, Masako

    Laminin is one of the components of the basement membrane and has diverse biological activities. Several functional peptides (EF1-EF5) are identified from LG4 modules of laminin alpha 1-5 chains. Thus, we perform conformation analysis of EF1 and EF2 using molecular dynamics simulations. In this study, we perform structure sampling with NPT ensemble (300 K, 1 bar). Our results show that EF1 peptide has β-sheet structure in water, and EF2 peptide does not have. Likewise, the EF2 peptide has unstable structure compared with the EF1 peptide in water.

  14. Complications of cataract surgery in patients with BPH treated with alpha 1A-blockers

    PubMed Central

    Dobrowolski, Dariusz; Wylegala, Edward

    2011-01-01

    The prevalence of benign prostate hyperplasia (BPH) and cataract increases with age. Both diseases may develop concomitantly and may affect almost 50% of elderly men as comorbidities. Cataract is treated surgically and it has been reported that there may be an association between use of alpha-blockers for BPH, particularly alpha1A-adrenergic receptor selective drugs, and complications of cataract surgery known as Intraoperative Floppy Iris Syndrome (IFIS). The article reviews literature published on this topic and provides recommendations on how to reduce incidence of iatrogenic IFIS or its severity and outcomes in patients with BPH. PMID:24578865

  15. Successful alpha-1 receptor blockade therapy in a toddler with infrequent and difficult voiding.

    PubMed

    Robson, William Lane M; Leung, Alexander K C

    2005-01-01

    A 3-year-old neurologically intact and behaviorally normal boy developed infrequent and difficult voiding subsequent to a soft tissue injury to the glans penis. Symptoms persisted for at least 9 months, and the course was complicated by diagnostic imaging evidence of a "markedly distended" bladder and a voiding diary that suggested elevated bladder volumes. Treatment with an alpha-1 receptor blocker normalized voiding within 24 hours. Discontinuation of the medication after 2 weeks resulted in recurrence of symptoms within 48 hours. Readministration of the medication resulted in prompt resolution of symptoms.

  16. Induced pluripotent stem cells model personalized variations in liver disease due to α1-antitrypsin deficiency

    PubMed Central

    Tafaleng, Edgar N.; Chakraborty, Souvik; Han, Bing; Hale, Pamela; Wu, Wanquan; Soto-Gutierrez, Alejandro; Feghali-Bostwick, Carol A.; Wilson, Andrew A.; Kotton, Darrell N.; Nagaya, Masaki; Strom, Stephen C.; Chowdhury, Jayanta R.; Stolz, Donna B.; Perlmutter, David H.; Fox, Ira J.

    2015-01-01

    In the classical form of α1-antitrypsin deficiency (ATD), aberrant intracellular accumulation of misfolded mutant α1-antitrypsin Z (ATZ) in hepatocytes causes hepatic damage by a gain-of-function, “proteotoxic” mechanism. While some ATD patients develop severe liver disease that necessitates liver transplantation, others with the same genetic defect completely escape this clinical phenotype. We investigated whether induced pluripotent stem cells (iPScs) from ATD individuals with or without severe liver disease could model these personalized variations in hepatic disease phenotypes. Patient-specific iPScs were generated from ATD patients and controls and differentiated into hepatocyte-like cells (iHeps) having many characteristics of hepatocytes. Pulse-chase and endoglycosidase H analysis demonstrate that the iHeps recapitulate the abnormal accumulation and processing of the ATZ molecule compared to the wild type AT molecule. Measurements of the fate of intracellular ATZ show a marked delay in the rate of ATZ degradation in iHeps from severe liver disease patients compared to those from no liver disease patients. Transmission electron microscopy showed dilated rER in iHeps from all individuals with ATD, not in controls, but globular inclusions that are partially covered with ribosomes were observed only in iHeps from individuals with severe liver disease. Conclusion These results provide definitive validation that iHeps model the individual disease phenotypes of ATD patients with more rapid degradation of misfolded ATZ and lack of globular inclusions in cells from patients who have escaped liver disease. The results support the concept that “proteostasis” mechanisms, such as intracellular degradation pathways, play a role in observed variations in clinical phenotype and show that iPScs can potentially be used to facilitate predictions of disease susceptibility for more precise and timely application of therapeutic strategies. PMID:25690322

  17. Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells

    PubMed Central

    Yusa, Kosuke; Rashid, S. Tamir; Strick-Marchand, Helene; Varela, Ignacio; Liu, Pei-Qi; Paschon, David E.; Miranda, Elena; Ordóñez, Adriana; Hannan, Nick; Rouhani, Foad Jafari; Darche, Sylvie; Alexander, Graeme; Marciniak, Stefan J.; Fusaki, Noemi; Hasegawa, Mamoru; Holmes, Michael C.; Di Santo, James P.; Lomas, David A.; Bradley, Allan; Vallier, Ludovic

    2011-01-01

    Human induced pluripotent stem cells (hIPSCs) represent a unique opportunity for regenerative medicine since they offer the prospect of generating unlimited quantities of cells for autologous transplantation as a novel treatment for a broad range of disorders1,2,3,4. However, the use of hIPSCs in the context of genetically inherited human disease will require correction of disease-causing mutations in a manner that is fully compatible with clinical applications3,5. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome6. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of hIPSCs. Here, we show that a combination of zinc finger nucleases (ZFNs)7 and piggyBac8,9 technology in hIPSCs can achieve bi-allelic correction of a point mutation (Glu342Lys) in the α1-antitrypsin (A1AT, also called SERPINA1) gene that is responsible for α1-antitrypsin deficiency (A1ATD). Genetic correction of hIPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle for the potential of combining hIPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies. PMID:21993621

  18. Perinatal lethal osteogenesis imperfecta in transgenic mice bearing an engineered mutant pro-alpha 1(I) collagen gene.

    PubMed

    Stacey, A; Bateman, J; Choi, T; Mascara, T; Cole, W; Jaenisch, R

    1988-03-10

    Substitutions of single glycine residues of alpha 1(I) collagen have previously been associated with the inherited disease osteogenesis imperfecta type II. Transgenic mice bearing a mutant alpha 1(I) collagen gene into which specific glycine substitutions have been engineered show a dominant lethal phenotype characteristic of the human disease, and demonstrate that as little as 10% mutant gene expression can disrupt normal collagen function.

  19. Method of using alpha-1 acid glycoprotein on T-cells as a marker for alzheimer's disease

    SciTech Connect

    Fudenberg, H.H.

    1989-01-31

    A method is described of diagnosing a dementia of the Alzheimer's type characterized by a change in the percentage of T-cells bearing surface membrane alpha-1 acid glycoprotein which comprises providing T-cells from a subject, determining the percentage of those T cells which bear surface membrane alpha-1 acid glycoprotein, and comparing that percentage of the percentage of T cells which bear the glycoprotein in a control, whereby the dementia is diagnosed.

  20. Insular cortex alpha1-adrenoceptors modulate the parasympathetic component of the baroreflex in unanesthetized rats.

    PubMed

    Alves, Fernando H F; Crestani, Carlos C; Resstel, Leonardo B M; Correa, Fernando M A

    2009-10-27

    The insular cortex (IC) has been reported to modulate the cardiac parasympathetic activity of the baroreflex in unanesthetized rats. However, which neurotransmitters are involved in this modulation is still unclear. In the present study, we evaluated the possible involvement of local IC-noradrenergic neurotransmission in modulating reflex bradycardiac responses. Bilateral microinjection of the selective alpha(1)-adrenoceptor antagonist WB4101 (15 nmol/100 nL), into the IC of male Wistar rats, increased the gain of reflex bradycardia in response to mean arterial pressure (MAP) increases evoked by intravenous infusion of phenylephrine. However, bilateral microinjection of equimolar doses of either the selective alpha(2)-adrenoceptor antagonist RX821002 or the non-selective beta-adrenoceptor antagonist propranolol into the IC did not affect the baroreflex response. No effects were observed in basal MAP or heart rate values after bilateral microinjection of noradrenergic antagonists into the IC, thus suggesting no tonic influence of IC-noradrenergic neurotransmission on resting cardiovascular parameters. In conclusion, these data provide evidence that local IC-noradrenergic neurotransmission has an inhibitory influence on baroreflex responses to blood pressure increase evoked by phenylephrine infusion through activation of alpha(1)-adrenoceptors. PMID:19686710

  1. Positive inotropic effects mediated by alpha 1 adrenoceptors in intact human subjects.

    PubMed

    Curiel, R; Pérez-González, J; Brito, N; Zerpa, R; Téllez, D; Cabrera, J; Curiel, C; Cubeddu, L

    1989-10-01

    The role of alpha 1-adrenergic receptors (adrenoceptors) on cardiac contractility was investigated in human subjects. The effect of methoxamine, a selective alpha-adrenoceptor agonist, and angiotensin II, on cardiac contractility was determined by means of noninvasive assessment of the slope of the end-systolic pressure (ESP)/end-systolic dimension (ESD) relationship. The slope (m) of this ratio was significantly higher with methoxamine (17.0; SD = 9.0 mm Hg/mm) than with angiotensin II (4.8; SD = 1.9 mm Hg/mm) (p less than 0.05). Slopes with methoxamine were higher when heart rates (HRs) were reflexly reduced, and were significantly diminished when reflex bradycardia was prevented by atropine (p less than 0.05) or atrial pacing (p less than 0.01). Previous treatment with propranolol did not modify m values with methoxamine (m = 15.5; SD = 4.4 mm Hg/mm). Phentolamine, given at peak methoxamine effect, did not consistently modify m values, resulting in an average slope not significantly different from that obtained with methoxamine alone. However, the addition of phentolamine did not cause an increase in ESDs at each level of ESP with respect to methoxamine. In the same subjects, infusion of phentolamine after angiotensin did not modify ESDs at comparable ESP levels. These findings suggest the existence of a positive inotropic effect mediated by alpha 1 adrenoceptors in the intact human heart.

  2. Functional analysis of {alpha}1,3/4-fucosyltransferase VI in human hepatocellular carcinoma cells

    SciTech Connect

    Guo, Qiya; Guo, Bin; Wang, Yingming; Wu, Jun; Jiang, Wenjun; Zhao, Shenan; Qiao, Shouyi; Wu, Yanhua

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Human FUT6 is up-regulated in HCC tissues. Black-Right-Pointing-Pointer Expression of FUT6 promotes G0/G1-S transition and cell growth. Black-Right-Pointing-Pointer FUT6 confers a growth advantage in vivo. Black-Right-Pointing-Pointer FUT6 suppresses p21 expression through modulating PI3K/Akt signaling. -- Abstract: The {alpha}1,3/4-fucosyltransferases (FUT) subfamily are key enzymes in cell surface antigen synthesis during various biological processes. A novel role of FUTs in tumorigenesis has been discovered recently, however, the underlying mechanism remains largely unknown. Here, we characterized FUT6, a member of {alpha}1,3/4-FUT subfamily, in human hepatocellular carcinoma (HCC). In HCC tissues, the expression levels of FUT6 and its catalytic product SLe{sup x} were significantly up-regulated. Overexpression of FUT6 in HCC cells enhanced S-phase cell population, promoted cell growth and colony formation ability. Moreover, subcutaneously injection of FUT6-overexpressing cells in nude mice promoted cell growth in vivo. In addition, elevating FUT6 expression markedly induced intracellular Akt phosphorylation, and suppressed the expression of the cyclin-dependent kinases inhibitor p21. Bath application of the PI3K inhibitor blocked FUT6-induced Akt phosphorylation, p21 suppression and cell proliferation. Our results suggest that FUT6 plays an important role in HCC growth by regulating the PI3K/Akt signaling pathway.

  3. Green tea modulates alpha(1)-adrenergic stimulated glucose transport in cultured rat cardiomyocytes.

    PubMed

    Angeloni, Cristina; Maraldi, Tullia; Ghelli, Anna; Rugolo, Michela; Leoncini, Emanuela; Hakim, Gabriele; Hrelia, Silvana

    2007-09-01

    alpha1-Adrenergic stimulation triggers glucose transport in the heart through the translocation of glucose transporter (GLUT) 1 and GLUT4 to plasma membranes, mediated by protein kinase C (PKC) isoforms. Evidence is emerging that dietary polyphenolic compounds may act not only as antioxidants but also by modulating PKC-mediated signaling. This study evaluated the ability of a green tea extract (GTE) to modulate alpha1-adrenoceptor-mediated glucose transport in rat cardiomyocytes. GTE supplementation decreased phenylephrine (PhE)-stimulated glucose uptake and GLUT4 recruitment. PhE stimulation activated PKC alpha, beta, delta, and epsilon, while GTE supplementation decreased the translocation of beta and delta isoforms, but not alpha and epsilon, supporting the notion that GTE directly affects PKC activation and is a beta and delta isoform-selective PKC inhibitor. Due to reactive oxygen species (ROS) involvement in pathological heart alterations, the observation that GTE is able to both inhibit effects originated by some PKC isoforms and counteract ROS deleterious effects could be important in the prevention/counteraction of these diseases.

  4. Possible role of alpha-1-microglobulin in mediating bacterial attachment to model surfaces.

    PubMed

    Wassall, M A; Santin, M; Peluso, G; Denyer, S P

    1998-06-01

    Urine proteins in the molecular weight range of 9-137 kDa deposit to an equal extent from pooled human urine onto glass (12.7 +/- 1.9 micrograms/cm) and polystyrene (11.8 +/- 1.8 micrograms/cm). Selective desorption of the proteins was achieved by washing with water or water/isopropanol mixtures. Irrespective of the washing process, proteins of molecular weight greater than 90 kDa remained associated with both surfaces while water washings alone removed most low molecular weight material. A 29 kDa protein, alpha-1-microglobulin, was removed from glass by water washing but required a 30% (v/v) isopropanol wash to desorb from polystyrene, implying attachment via hydrophobic bonding. The adhesion to polystyrene surfaces of Pseudomonas aeruginosa B4, a clinical isolate from a urinary tract infection (UTI), was strongly associated with the presence of alpha-1-microglobulin, which may be acting as a mediator of bacterial adhesion.

  5. Doxazosin inhibits proliferation and migration of human vascular smooth-muscle cells independent of alpha1-adrenergic receptor antagonism.

    PubMed

    Hu, Z W; Shi, X Y; Hoffman, B B

    1998-06-01

    Proliferation and migration of vascular smooth-muscle cells (VSMCs), stimulated by a variety of growth factors, play a critical role in the pathogenesis of vascular diseases. We found unexpectedly that doxazosin, an alpha1-adrenergic-receptor antagonist, inhibits serum-stimulated proliferation of cultured human VSMCs. Subsequent experiments systematically investigated inhibitory effects of doxazosin on mitogenesis stimulated in VSMCs by platelet-derived growth factor (PDGF), epidermal growth factor, and G protein-coupled receptor agonists thrombin and angiotensin II. Doxazosin attenuated the stimulation of DNA synthesis for each of these ligands with median inhibitory concentrations (IC50s) from 0.3 to 1 microM. PDGF-AB (1 nM) increased cell number; doxazosin inhibited this response by 70-80%. Prazosin, a related alpha1-receptor antagonist, had similar but less potent effects on inhibiting mitogenesis in these cells. Doxazosin and prazosin inhibited PDGF-AB-stimulated and insulin-like growth factor (IGF-I)-stimulated migration of VSMCs by approximately 40-50%. These effects of doxazosin were likely unrelated to alpha1-receptor blockade because pretreatment of cells with phenoxybenzamine, an irreversible alpha1 antagonist, did not change the capacity of doxazosin to inhibit of PDGF-stimulated mitogenesis. Also, doxazosin inhibited PDGF-stimulated DNA synthesis in NIH 3T3 cells, which do not express alpha1 receptors. These results suggest that doxazosin is a potent inhibitor of VSMC proliferation and migration through a mechanism unrelated to alpha1-receptor antagonism.

  6. Inhibition of laminin alpha 1-chain expression leads to alteration of basement membrane assembly and cell differentiation

    PubMed Central

    1996-01-01

    The expression of the constituent alpha 1 chain of laminin-1, a major component of basement membranes, is markedly regulated during development and differentiation. We have designed an antisense RNA strategy to analyze the direct involvement of the alpha 1 chain in laminin assembly, basement membrane formation, and cell differentiation. We report that the absence of alpha 1-chain expression, resulting from the stable transfection of the human colonic cancer Caco2 cells with an eukaryotic expression vector comprising a cDNA fragment of the alpha 1 chain inserted in an antisense orientation, led to (a) an incorrect secretion of the two other constituent chains of laminin-1, the beta 1/gamma 1 chains, (b) the lack of basement membrane assembly when Caco2-deficient cells were cultured on top of fibroblasts, assessed by the absence of collagen IV and nidogen deposition, and (c) changes in the structural polarity of cells accompanied by the inhibition of an apical digestive enzyme, sucrase-isomaltase. The results demonstrate that the alpha 1 chain is required for secretion of laminin-1 and for the assembly of basement membrane network. Furthermore, expression of the laminin alpha 1-chain gene may be a regulatory element in determining cell differentiation. PMID:8609173

  7. Functional analysis of developmentally regulated chromatin-hypersensitive domains carrying the alpha 1-fetoprotein gene promoter and the albumin/alpha 1-fetoprotein intergenic enhancer.

    PubMed Central

    Bernier, D; Thomassin, H; Allard, D; Guertin, M; Hamel, D; Blaquière, M; Beauchemin, M; LaRue, H; Estable-Puig, M; Bélanger, L

    1993-01-01

    During liver development, the tandem alpha 1-fetoprotein (AFP)/albumin locus is triggered at the AFP end and then asymmetrically enhanced; this is followed by autonomous repression of the AFP-encoding gene. To understand this regulation better, we characterized the two early developmental stage-specific DNase I-hypersensitive (DH) sites so far identified in rat liver AFP/albumin chromatin: an intergenic DH-enhancer site and the AFP DH-promoter site. Mutation-transfection analyses circumscribed the DH-enhancer domain to a 200-bp DNA segment stringently conserved among species. Targeted mutations, DNA-protein-binding assays, and coexpression experiments pinpointed C/EBP as the major activatory component of the intergenic enhancer. Structure-function relationships at the AFP DH-promoter site defined a discrete glucocorticoid-regulated domain activated cooperatively by HNF1 and a highly specific AFP transcription factor, FTF, which binds to a steroid receptor recognition motif. The HNF1/FTF/DNA complex is deactivated by glucocorticoid receptors or by the ubiquitous factor NF1, which eliminates HNF1 by competition at an overlapping, high-affinity binding site. We propose that the HNF1-NF1 site might serve as a developmental switch to direct autonomous AFP gene repression in late liver development. We also conclude that the intergenic enhancer is driven by C/EBP alpha primarily to fulfill albumin gene activation functions at early developmental stages. Factor FTF seems to be the key regulator of AFP gene-specific functions in carcinoembryonic states. Images PMID:7680097

  8. Introduction of the human pro. cap alpha. 1(I) collagen gene into pro. cap alpha. 1(I)-deficient Mov-13 mouse cells leads to formation of functional mouse-human hybrid type I collagen

    SciTech Connect

    Schnieke, A.; Dziadek, M.; Bateman, J.; Mascara, T.; Harbers, K.; Gelinas, R.; Jaenisch, R.

    1987-02-01

    The Mov-13 mouse strain carries a retroviral insertion in the pro..cap alpha..1(I) collagen gene that prevents transcription of the gene. Cell lines derived from homozygous embryos do not express type I collagen although normal amounts of pro..cap alpha..2 mRNA are synthesized. The authors have introduced genomic clones of either the human or mouse pro..cap alpha..1(I) collagen gene into homozygous cell lines to assess whether the human or mouse pro..cap alpha..1(I) chains can associate with the endogenous mouse pro..cap alpha..2(I) chain to form stable type I collagen. The human gene under control of the simian virus 40 promoter was efficiently transcribed in the transfected cells. Protein analyses revealed that stable heterotrimers consisting of two human ..cap alpha..1 chains and one mouse ..cap alpha..2 chain were formed and that type I collagen was secreted by the transfected cells at normal rates. However, the electrophoretic migration of both ..cap alpha..1(I) and ..cap alpha..2(I) chains in the human-mouse hybrid molecules were retarded, compared to the ..cap alpha..(I) chains in control mouse cells. Inhibition of the posttranslational hydroxylation of lysine and proline resulted in comigration of human and mouse ..cap alpha..1 and ..cap alpha..2 chains, suggesting that increased posttranslational modification caused the altered electrophoretic migration in the human-mouse hybrid molecules. Amino acid sequence differences between the mouse and human ..cap alpha.. chains may interfere with the normal rate of helix formation and increase the degree of posttranslational modifications similar to those observed in patients with lethal perinatal osteogenesis imperfecta. The Mov-13 mouse system should allow the authors to study the effect specific mutations introduced in transfected pro..cap alpha..1(I) genes have on the synthesis, assembly, and function of collagen I.

  9. Introduction of the human pro alpha 1(I) collagen gene into pro alpha 1(I)-deficient Mov-13 mouse cells leads to formation of functional mouse-human hybrid type I collagen.

    PubMed Central

    Schnieke, A; Dziadek, M; Bateman, J; Mascara, T; Harbers, K; Gelinas, R; Jaenisch, R

    1987-01-01

    The Mov-13 mouse strain carries a retroviral insertion in the pro alpha 1(I) collagen gene that prevents transcription of the gene. Cell lines derived from homozygous embryos do not express type I collagen although normal amounts of pro alpha 2 mRNA are synthesized. We have introduced genomic clones of either the human or mouse pro alpha 1(I) collagen gene into homozygous cell lines to assess whether the human or mouse pro alpha 1(I) chains can associate with the endogenous mouse pro alpha 2(I) chain to form stable type I collagen. The human gene under control of the simian virus 40 promoter was efficiently transcribed in the transfected cells. Protein analyses revealed that stable heterotrimers consisting of two human alpha 1 chains and one mouse alpha 2 chain were formed and that type I collagen was secreted by the transfected cells at normal rates. However, the electrophoretic migration of both alpha 1(I) and alpha 2(I) chains in the human-mouse hybrid molecules were retarded, compared to the alpha (I) chains in control mouse cells. Inhibition of the posttranslational hydroxylation of lysine and proline resulted in comigration of human and mouse alpha 1 and alpha 2 chains, suggesting that increased posttranslational modification caused the altered electrophoretic migration in the human-mouse hybrid molecules. Amino acid sequence differences between the mouse and human alpha chains may interfere with the normal rate of helix formation and increase the degree of posttranslational modifications similar to those observed in patients with lethal perinatal osteogenesis imperfecta. The Mov-13 mouse system should allow us to study the effect specific mutations introduced in transfected pro alpha 1(I) genes have on the synthesis, assembly, and function of collagen I. Images PMID:3468512

  10. Alpha 1-proteinase inhibitor is more sensitive to inactivation by cigarette smoke than is leukocyte elastase

    SciTech Connect

    Janoff, A.; Dearing, R.

    1982-10-01

    Aqueous solutions of gas phase cigarette smoke were incubated with pure human leukocyte elastase or with crude human leukocyte granule extract, and the effects on enzyme activity were determined using a synthetic amide substrate. Simultaneously, the same smoke solutions were incubated with 10% human serum under identical conditions, and the effects on serum inhibition of purified or crude leukocyte elastase were similarly measured. In addition, aqueous solutions of unfractionated cigarette smoke were incubated with leukocyte elastase or serum, and the abilities of the smoke-treated enzyme to digest elastin and of the smoke-treated serum to inhibit elastin digestion were determined. Both experimental protocols showed that serum elastase-inhibiting capacity (primarily caused by alpha 1-proteinase inhibitor) is more susceptible to inactivation by aqueous solutions of cigarette smoke than is leukocyte elastase, suggesting that elastase inhibition (rather than elastase activity) may be predominantly suppressed by cigarette smoke inhalation in vivo.

  11. (-)-Discretamine, a selective alpha 1D-adrenoceptor antagonist, isolated from Fissistigma glaucescens.

    PubMed

    Ko, F N; Guh, J H; Yu, S M; Hou, Y S; Wu, Y C; Teng, C M

    1994-08-01

    1. The selectivity of (-)-discretamine for alpha 1-adrenoceptor subtypes was investigated by use of functional and binding studies in rat vas deferens, spleen and aorta, and in cultured DDT1MF-2 and A10 cells. 2. In prostatic portions of rat vas deferens, the competitive antagonists (-)-discretamine, 5-methylurapidil (5-MU) and prazosin inhibited contractions to noradrenaline (NA) with pA2 values of 6.21, 8.71 and 9.27, respectively. The irreversible antagonist, chloroethylclonidine (CEC, 100 microM) failed to affect contractions to NA while nifedipine (1 microM) blocked them almost completely. 3. In rat spleen, the competitive antagonists (-)-discretamine, 5-MU and prazosin inhibited contractions to phenylephrine with pA2 values of 6.44, 7.19 and 9.45, respectively. CEC (100 microM) significantly reduced the maximum contraction to phenylephrine while nifedipine (1 microM) did not affect it. 4. In rat aorta, the competitive antagonists (-)-discretamine, 5-MU and prazosin inhibited contractions to NA with pA2 values of 7.60, 8.00 and 9.40, respectively. CEC also antagonized the contractions to NA in a competitive manner with a pA2 value of 6.10. 5. The specific binding of [3H]-prazosin to DDT1MF-2 and A10 cells was concentration-dependent and saturated at 3-5 nM with KD values of 0.24 +/- 0.02 and 0.20 +/- 0.02 nM, respectively. (-)-Discretamine,5-MU, CEC and prazosin inhibited specific [3H]-prazosin binding to DDTIMF-2 and AlO cells in a concentration-dependent manner with ICso values of 390.8 +/- 20.6, 43.6 +/- 3.9, 200.0 +/- 30.0 and 0.8 +/- 0.1 nM, respectively in DDTIMF-2 cells, and 25.0 +/- 3.2, 8.6 +/- 1.4, 1000.0 +/- 30.8 and 0.52 +/- 0.03 nM, respectively in AlO cells.6. Pretreatment of Al0 cells with CEC (10 MicroM) for 30 min and then washed out thoroughly, reduced specific [3H]-prazosin binding by 30%. The CEC-insensitive [3H]-prazosin binding was inhibited by(-)-discretamine with an IC50 value of 7.0 +/- 0.3 nM.7. 5-MU (100 nM), CEC (1 MicroM) and

  12. Radiation dosimetry of iodine-123 HEAT, an alpha-1 receptor imaging agent

    SciTech Connect

    Thomas, K.D.; Greer, D.M.; Couch, M.W.; Williams, C.M.

    1987-11-01

    Biologic distribution data in the rat were obtained for the alpha-1 adrenoceptor imaging agent (+/-) 2-(beta-(iodo-4-hydroxyphenyl)ethylaminomethyl)tetralone (HEAT) labeled with (/sup 123/I). The major excretory routes were through the liver (67%) and the kidney (33%). Internal radiation absorbed dose estimates to nine source organs, total body, the GI tract, gonads, and red bone marrow were calculated for the human using the physical decay data for (/sup 123/I). The critical organ was found to be the lower large intestine, receiving 1.1 rad per mCi of (/sup 123/I)HEAT administered. The total-body dose was found to be 58 mrad per mCi.

  13. Activity of alpha-1, 4-glucosidase in furazolidone-induced glycogenosis.

    PubMed

    Czarnecki, C M; Salam, A; Caldwell, R; Jankus, E F

    1978-01-01

    Furazolidone (FZ) at 700 and 800 p.p.m. was added to feed mixtures fed turkey poults two and three weeks posthatching, respectively, to induce acute experimental cardiomyopathy. Poults in the control pen received the same ration but without FZ. From EKG data obtained at 2, 4, and 5 weeks of age, control unaffected and experimental affected poults were selected for sacrifice. Poults were sacrificed by cervical dislocation and appropriate samples of hepatic tissue were removed for assays of activity of alpha-1, 4-glucosidase. Results indicate that enzyme activity in affected FZ-treated poults is similar to that in unaffected control poults. Lack of significant differences in activity of this lysosomal enzyme suggests that FZ-induced glycogenosis may be related to the adult form of idiopathic generalized glucogenosis, the etiology of which remains unidentified. PMID:353772

  14. Relationship between alpha 1-adrenergic receptor occupancy and response in BC3H-1 muscle cells

    SciTech Connect

    Brown, R.D.; Berger, K.D.; Taylor, P.

    1987-07-01

    The relationship between alpha 1-adrenergic receptor occupancy by agonists or antagonists and the regulation of intracellular Ca/sup 2 +/ was examined. Receptor occupancy was measured using the antagonist (/sup 3/H)prazosin and correlated with agonist-elicited /sup 45/Ca/sup 2 +/ fluxes. The agonists epinephrine (E), norepinephrine (NE), and phenylephrine (PE) coordinately activated Ca/sup 2 +/ efflux, reflecting a substantial mobilization of intracellular Ca/sup 2 +/, as well as a smaller /sup 45/Ca/sup 2 +/ influx. The agonist concentration dependences for influx and efflux were similar, with the order of potency expected for alpha 1 receptors (E greater than or equal to NE greater than PE). To determine the relationship between receptor occupancy and response, the slowly dissociating antagonist prazosin was used to inactivate specified fractions of the receptor population. A linear relationship was observed between the remaining activatable receptors and residual /sup 45/Ca/sup 2 +/ efflux elicited by E or NE, except at saturating agonist concentrations where some curvature was observed. Moreover, the concentration dependence for agonist-elicited /sup 45/Ca/sup 2 +/ efflux was shifted toward slightly higher concentrations of E or NE following prazosin inactivation. These results suggest the presence of a modest receptor reserve which is revealed by E or NE, but not by PE. Agonist occupation was measured over the same interval as receptor activation by competition with the initial rate of (/sup 3/H)prazosin association. All three agonists exhibited the major fraction of receptor occupation over the same concentration ranges required for the functional response. Exposure of receptors to specified agonist concentrations for 30 min had little effect on the number of receptors or their ligand affinities, whereas a 2.5-hr exposure to agonist decreased apparent agonist affinity as well as the number of receptors recognized by (/sup 3/H)prazosin.

  15. Binding of oxprenolol and propranolol to serum, albumin and alpha 1-acid glycoprotein in man and other species.

    PubMed

    Belpaire, F M; Braeckman, R A; Bogaert, M G

    1984-07-01

    Species differences in binding of basic drugs have only occasionally been studied and we have therefore measured the binding of the beta-adrenergic blockers oxprenolol and propranolol in (1) serum of healthy humans, dogs, rats and rabbits and of rabbits with experimental arthritis, (2) a solution of albumin of these species and (3) a solution of human alpha 1-AGP. In humans, dogs, rats and arthritic rabbits, binding of oxprenolol and propranolol was much higher in serum than in albumin solution; in healthy rabbits serum binding was very low and not different from albumin binding. For both drugs, concentration-dependency was seen in serum of dogs, humans and rats and of arthritic rabbits; a similar concentration-dependency was found for human alpha 1-AGP solution, but not for human albumin and for serum of healthy rabbits. Tris (2-butoxyethyl)-phosphate (TBEP), a known displacer of drugs from alpha 1-AGP in humans, decreased binding in serum of all species except the rabbit. For both beta-blockers, species differences in capacity constants were found; species differences in affinity constants were present only for propranolol. These results suggest that in humans, dog and rat, but much less in rabbits, oxprenolol and propranolol bind mainly to alpha 1-AGP and that binding to alpha 1-AGP is more important for oxprenolol than for propranolol. PMID:6743355

  16. [Uroselectivity of alpha-1 antagonism in the treatment of benign prostatic hypertrophy: on the pharmacologic concept of the clinical approach].

    PubMed

    Jolliet, P; Bourin, M

    1998-01-01

    Benign prostatic hyperplasia is the most common cause of voiding dysfunction in men. It becomes symptomatic from the fifth decade of life and needs treatment in 50 per cent of patients. Hyperplastic prostatic tissue and the smooth involuntary sphincter have a high density of alpha 1-adrenoceptors, thus alpha 1-blockers can decrease sphincter tone and reduce the tension exerted by the prostatic muscular component. Attempts have been made to find alpha 1-antagonists that have a selective effect on the prostate (alfuzosin), are long acting (tamsulosin, terazosin, doxazosin) or present specificity on the alpha 1A prostatic adrenoceptors (tamsulosin), in order to maintain efficacy without affecting blood pressure. Finasteride, a 5 alpha-reductase inhibitor without hypotensive side-effect may be more effective in men with a predominantly glandular component to their benign hyperplasia or with very large prostate glands, but has a longer onset of action and produces more adverse sexual effects. Thus, alpha-1 antagonists can be considered as an appropriate treatment option in patients with troublesome symptoms of BPH and who have not developed serious complications indicating surgery.

  17. Combined effect of fluconazole and thymosin alpha 1 on systemic candidiasis in mice immunosuppressed by morphine treatments.

    PubMed Central

    di Francesco, P; Gaziano, R; Casalinuovo, I A; Belogi, L; Palamara, A T; Favalli, C; Garaci, E

    1994-01-01

    Treatment of systemic infection with Candida albicans with a combination of an antifungal agent (i.e. fluconazole) and a thymus-derived immunostimulant (i.e. thymosin alpha 1 (T alpha 1)) in mice immunosuppressed by morphine treatments was investigated. In normal mice, fluconazole given after infection with 10(6) C. albicans cells was more effective than in mice treated with morphine. Combination treatment with fluconazole and T alpha 1 prolonged survival and reduced the fungal burden in the kidneys of immunosuppressed mice. We also investigated the influence of this combined treatment on killing properties of polymorphonuclear leucocytes (PMN) and natural killer (NK) cell activity, inhibited by morphine administrations. Treatment with T alpha 1 or fluconazole as single agents promoted a recovery of normal NK cell activity and intracellular killing of C. albicans by PMN, while the combination significantly increased both of these responses, probably through the modulation of lymphokine production. Our data suggest that the additive effect of T alpha 1 and fluconazole is due to a direct antifungal action and activation of the immunocompetence. PMID:8082290

  18. Directing membrane chromatography to manufacture α1-antitrypsin from human plasma fraction IV.

    PubMed

    Fan, Jinxin; Luo, Jianquan; Song, Weijie; Chen, Xiangrong; Wan, Yinhua

    2015-12-01

    The surging demand for plasma proteins, mainly driven by the growing market and the development of new therapeutic indications, is promoting manufacturers to improve the throughput of plasma proteins. Due to the inherent convective mass transfer, membrane chromatography has been proved to be an efficient approach for extracting a small amount of target proteins from large-volume feed. In this study, α1-antitrypsin (AAT) was extracted from human plasma fraction IV by a two-step membrane chromatography. An anion-exchange membrane chromatography (AEMC) was used to capture the plasma proteins in bind/elute mode, and the obtained effluent was further polished by a hydrophobic interaction membrane chromatography (HIMC) in flow-through mode. Under optimal conditions, the recovery and purity of AAT achieved 87.0% and 0.58 AAT/protein (g/g) by AEMC, respectively. After the precise polishing by HIMC, the purity of AAT was 1.22 AAT/protein (g/g). The comparison results showed that membrane chromatography outperformed column chromatography in both steps because of its high throughput. This two-step membrane chromatography could obtain an AAT recovery of 83.3% and an activity recovery of 91.4%. The outcome of this work not only offers an alternative process for protein purification from plasma, but also provides guidelines for manufacturing product from a large-volume feed with multi-components by membrane chromatography.

  19. Altered native stability is the dominant basis for susceptibility of α1-antitrypsin mutants to polymerization

    PubMed Central

    Irving, James A.; Haq, Imran; Dickens, Jennifer A.; Faull, Sarah V.; Lomas, David A.

    2014-01-01

    Serpins are protease inhibitors whose most stable state is achieved upon transition of a central 5-stranded β-sheet to a 6-stranded form. Mutations, low pH, denaturants and elevated temperatures promote this transition, which can result in a growing polymer chain of inactive molecules. Different types of polymer are possible, but, experimentally only heat has been shown to generate polymers in vitro consistent with ex vivo pathological specimens. Many mutations that alter the rate of heat-induced polymerization have been described, but interpretation is problematic because discrimination is lacking between the effect of global changes in native stability and specific effects on structural mechanism. We show that the temperature midpoint (Tm) of thermal denaturation reflects the transition of α1-antitrypsin to the polymerization intermediate, and determine the relationship with fixed-temperature polymerization half-times (t0.5) in the presence of stabilizing additives [TMAO (trimethylamine N-oxide), sucrose and sodium sulfate], point mutations and disulfide bonds. Combined with a retrospective analysis of 31 mutants characterized in the literature, the results of the present study show that global changes to native state stability are the predominant basis for the effects of mutations and osmolytes on heat-induced polymerization, summarized by the equation: ln(t0.5,mutant/t0.5,wild-type)=0.34×ΔTm. It is deviations from this relationship that hold key information about the polymerization process. PMID:24552432

  20. Regulation of autophagy by α1-antitrypsin: "a foe of a foe is a friend".

    PubMed

    Shapira, Michal G; Khalfin, Boris; Lewis, Eli C; Parola, Abraham H; Nathan, Ilana

    2014-10-27

    Autophagy is involved in both the cell protective and the cell death process but its mechanism is largely unknown. The present work unravels a novel intracellular mechanism by which the serpin α1-antitrypsin (AAT) acts as a novel negative regulator of autophagic cell death. For the first time, the role of intracellularly synthesized AAT, other than in liver cells, is demonstrated. Autophagic cell death was induced by N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and tamoxifen. By utilizing a fluorescently tagged TPCK analog, AAT was "fished out" (pulled out) as a TPCK intracellular protein target. The interaction was further verified by competition binding experiments. Both inducers caused downregulation of AAT expression associated with activation of trypsin-like proteases. Furthermore, silencing AAT by siRNA induced autophagic cell death. Moreover, AAT administration to cultured cells prevented autophagic cell death. This new mechanism could have implications in the treatment of diseases by the regulation of AAT levels in which autophagy has a detrimental function. Furthermore, the results imply that the high synthesis of endogenous AAT by cancer cells could provide a novel resistance mechanism of cancer against autophagic cell death.

  1. Selective increase of alpha 1-adrenoceptors and muscarinic cholinergic receptors in rat cerebral cortex after chronic haloperidol.

    PubMed

    Pazo, J H; Levi de Stein, M; Jerusalinsky, D; Novas, M L; Raskovsky, S; Tumilasci, O R; Medina, J H; De Robertis, E

    1987-06-30

    The effect of chronic administration of haloperidol on alpha 1-, alpha 2-, and beta-adrenoceptors, cholinergic muscarinic, GABAA and benzodiazepine receptors in the cerebral cortex of the rat was investigated. Doses of 0.3 and 2 mg/kg of haloperidol during 7 days increased markedly the density of alpha 1-adrenoceptors without changes in affinity. The alpha 2- and beta-adrenoceptors were not modified after neuroleptic administration. The number of muscarinic receptors were also increased after haloperidol treatment (2 mg/kg/day). However, the GABAA and benzodiazepine binding sites remained unchanged. In the brainstem an increment in the alpha 1-, but not the beta-adrenoceptors was observed. The well known increase in the dopamine receptors in the striatum was confirmed. These observations demonstrate a multireceptor effect of haloperidol in the cerebral cortex.

  2. Apoptosis induction by doxazosin and other quinazoline alpha1-adrenoceptor antagonists: a new mechanism for cancer treatment?

    PubMed

    Kyprianou, Natasha; Vaughan, Taylor B; Michel, Martin C

    2009-12-01

    Doxazosin and related, quinazoline-based alpha(1)-adrenoceptor antagonists can induce apoptosis in prostate and various other normal, benign, smooth muscle, endothelial and malignant cells. Such apoptosis-inducing effects occur independently of alpha(1)-adrenoceptor antagonism and typically require much high concentrations than those required for receptor occupancy. Several studies have invested efforts towards the elucidation of the molecular mechanisms underlying doxazosin-induced apoptosis. These include various tumor cells, cardiomyocytes, endothelial cells and bladder smooth muscle cells. While the high concentrations of doxazosin required to induce apoptosis challenge the use of this and related drugs for clinical optimization of apoptosis induction, such quinazoline structure may represent chemical starting points to develop more potent apoptosis-inducing agents free of alpha(1)-adrenoceptor antagonistic action and suitable for cancer treatment with minimal and well-tolerated side effects.

  3. Functional evidence of alpha1D-adrenoceptors in the vasculature of young and adult spontaneously hypertensive rats.

    PubMed

    Villalobos-Molina, R; López-Guerrero, J J; Ibarra, M

    1999-04-01

    The role of alpha1D-adrenoceptors in the vasculature of spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rats (WKY), of different ages was assessed in pithed rats by the use of the selective alpha1D-adrenoceptor antagonist BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl]-8-azaspiro [4.5]decane-7,9-dione dihydrochloride). BMY 7378 displaced the pressor effect of phenylephrine in young pre-hypertensive pithed SHR rats, but produced no effect in young WKY rats (dose ratio of 3.4 and 1.6, respectively), while in adult rats BMY 7378 produced a greater shift in the phenylephrine response curve than in younger animals (dose ratio of 3.2 and 6.2 in WKY and SHR, respectively). The presence of alpha1D-adrenoceptors in the vasculature of pre-hypertensive rats, suggests its role in the pathogenesis/maintenance of increased blood pressure. PMID:10323583

  4. A possible structural determinant of selectivity of boldine and derivatives for the alpha 1A-adrenoceptor subtype.

    PubMed Central

    Madrero, Y.; Elorriaga, M.; Martinez, S.; Noguera, M. A.; Cassels, B. K.; D'Ocon, P.; Ivorra, M. D.

    1996-01-01

    1. The selectivity of action of boldine and the related aporphine alkaloids, predicentrine (9-O-methylboldine) and glaucine (2,9-O-dimethylboldine) and alpha 1-adrenoceptor subtypes was studied by examining [3H]-prazosin competition binding in rat cerebral cortex. WB 4101 and benoxathian were used as selective alpha 1A-adrenoceptor antagonists. 2. In the competition experiments [3H]-prazosin (0.2 nM) binding was inhibited by WB 4101 and benoxathian. The inhibition curves displayed shallow slopes which could be subdivided into high and low affinity components (pKi = 9.92 and 8.29 for WB 4101, 9.35 and 7.94 for benoxathian). The two antagonists recognized approximately 37% of the sites with high affinity from among the total [3H]-prazosin specific binding sites. 3. Boldine, predicentrine and glaucine also competed for [3H]-prazosin (0.2 nM) binding with shallow and biphasic curves recognizing 30-40% of the sites with high affinity. Drug affinities (pKi) at the high and low affinity sites were, 8.31 and 6.50, respectively, for boldine, 8.13 and 6.39 for predicentrine, and 7.12 and 5.92 for glaucine. The relative order of selectivity for alpha 1A-adrenoceptors was boldine (70 fold alpha 1A-selective) = predicentrine (60 fold, alpha 1A-selective) > glaucine (15 fold, alpha 1A-selective). 4. Pretreatment of rat cerebral cortex membranes with chloroethylclonidine (CEC, 10 microM) for 30 min at 37 degrees C followed by thorough washing out reduced specific [3H]-prazosin binding by approximately 70%. The CEC-insensitive [3H]-prazosin binding was inhibited by boldine monophasically (Hill slope = 0.93) with a single pKi value (7.76). 5. These results suggest that whereas the aporphine structure shared by these alkaloids is responsible for their selectively of action for the alpha 1A-adrenoceptor subtype in rat cerebral cortex, defined functional groups, namely the 2-hydroxy function, induces a significant increase in alpha 1A-subtype selectivity and affinity. PMID:8982502

  5. Pharmacological characterization of alpha 1-adrenoceptor subtypes in rat heart: a binding study.

    PubMed Central

    Noguchi, H; Muraoka, R; Kigoshi, S; Muramatsu, I

    1995-01-01

    1. The alpha 1-adrenoceptor subtypes of rat heart were characterized in binding experiments performed with [3H]-prazosin as the radiolabel. The specific binding to the alpha 1-adrenoceptors was determined with 0.3 microM prazosin, because phentolamine (10 microM) was insufficient to inhibit completely the specific binding of high concentrations of [3H]-prazosin. 2. In saturation experiments, [3H]-prazosin bound to two distinct affinity sites (pKD = 10.39 and 8.19). The proportion of the low affinity sites was approximately 84% of total specific binding. Membranes pretreated with chloroethylclonidine (CEC, 10 microM) also showed two distinct affinity sites for [3H]-prazosin, although the maximum numbers of high and low affinity sites were reduced by 86 and 64%, respectively. 3. In competition experiments, [3H]-prazosin (100 pM) binding was inhibited by WB4101 (2-(2,6-dimethoxy-phenoxyethyl)aminomethyl-1,4-benzodioxane) and 5-methylurapidil. The inhibition curves displayed shallow slopes which could be subdivided into high and low affinity components (pKi = 10.43 and 8.36 for WB4101, 8.62 and 6.61 for 5-methylurapidil). However, unlabelled prazosin or HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)-ethyl)amin o) propyl)benzeneacetonitrile fumarate) competed for [3H]-prazosin binding monophasically (pKi = 10.34 and 8.28, respectively). In CEC-pretreated membranes, prazosin, WB4101, 5-methylurapidil and HV723 antagonized the [3H]-prazosin (100 pM) binding monophasically (pKi = 9.70, 9.56, 8.60 and 8.82, for each antagonist). 4. On the other hand, 1000 pM [3H]-prazosin binding was inhibited by unlabelled prazosin biphasically (pKi = 10.49 and 8.49). HV723 did not discriminate both prazosin-high and low affinity sites (pKi = 8.18).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7780636

  6. Molecular cloning of the alpha-1 subunit of an omega-conotoxin-sensitive calcium channel.

    PubMed Central

    Dubel, S J; Starr, T V; Hell, J; Ahlijanian, M K; Enyeart, J J; Catterall, W A; Snutch, T P

    1992-01-01

    Of the four major types of Ca channel described in vertebrate cells (designated T, L, N, and P), N-type Ca channels are unique in that they are found specifically in neurons, have been correlated with control of neurotransmitter release, and are blocked by omega-conotoxin, a neuropeptide toxin isolated from the marine snail Conus geographus. A set of overlapping cDNA clones were isolated and found to encode a Ca channel alpha-1 subunit, designated rbB-I. Polyclonal antiserum generated against a peptide from the rbB-I sequence selectively immunoprecipitates high-affinity 125I-labeled omega-conotoxin-binding sites from labeled rat forebrain membranes. PCR analysis shows that, like N-type Ca channels, expression of rbB-I is limited to the nervous system and neuronally derived cell lines. This brain Ca channel may mediate the omega-conotoxin-sensitive Ca influx required for neurotransmitter release at many synapses. Images PMID:1317580

  7. Cell type-specific transcriptional regulation of the gene encoding importin-{alpha}1

    SciTech Connect

    Kamikawa, Yasunao; Yasuhara, Noriko; Yoneda, Yoshihiro

    2011-08-15

    Importin-{alpha}1 belongs to a receptor family that recognizes classical nuclear localization signals. Encoded by Kpna2, this receptor subtype is highly expressed in mouse embryonic stem (ES) cells. In this study, we identified a critical promoter region in Kpna2 and showed that the expression of this gene is differentially regulated in ES cells and NIH3T3 cells. Conserved CCAAT boxes are required for Kpna2 promoter activity in both ES and NIH3T3 cells. Interestingly, deletion of the region from nucleotide position - 251 to - 179 bp resulted in a drastic reduction in Kpna2 transcriptional activity only in ES cells. This region contains Krueppel-like factor (Klf) binding sequences and is responsible for transactivation of the gene by Klf2 and Klf4. Accordingly, endogenous Kpna2 mRNA levels decreased in response to depletion of Klf2 and Klf4 in ES cells. Our results suggest that Klf2 and Klf4 function redundantly to drive high level of Kpna2 expression in ES cells. -- Research Highlights: {yields} We showed the cell type-specific transcriptional regulation of Kpna2 encoding importin-al. {yields} NF-Y binds the CCAAT boxes to activate Kpna2 transcription in NIH3T3 cells. {yields} Klf2 and Klf4 redundantly activate the expression of Kpna2 in ES cells.

  8. Correlation between phosphatidylinositol labeling and contraction in rabbit aorta: effect of alpha-1 adrenergic activation

    SciTech Connect

    Villalobos-Molina, R.; Uc, M.; Hong, E.; Garcia-Sainz, J.A.

    1982-07-01

    Activation of rabbit aortic strips with alpha adrenergic agonists increased the labeling (with (/sup 32/P)Pi) of phosphatidylinositol (PI) and phosphatidic acid and contracted the vascular preparations in dose-related fashion. Epinephrine, norepinephrine and methoxamine produced maximal effects, whereas clonidine behaved as partial agonist and B-HT 933 (2-amino-6-ethyl-4,5,7,8-tetrahydro-6H-oxazole-(5,4-d) azepin dihydrochloride) was almost without activity in the two experimental models used. Phenylephrine was a full agonist in producing contraction, but failed to elicit the maximal increase in PI labeling. The EC50 values to produce contraction of aortic strips were lower for all agonists than those required to increase the incorporation of radioactive phosphate into PI, but there was a good correlation between the two sets of data. The increased PI labeling and contraction of aortic strips induced by epinephrine were antagonized by prazosin and yohimbine in dose-related fashion, but the first alpha blocker was about three orders of magnitude more potent than the second in antagonizing the two effects. The present results indicate that both stimulation of PI labeling and contraction are mediated through activation of alpha-1 adrenoceptors in rabbit aorta.

  9. Alpha 1-adrenergic stimulation of phosphatidylinositol turnover and respiration of brown fat cells

    SciTech Connect

    Mohell, N.; Wallace, M.; Fain, J.N.

    1984-01-01

    The alpha-adrenergic agonist phenylephrine (in the presence of the beta-adrenergic antagonist alprenolol) stimulated respiration and incorporation of (/sup 3/H)glycerol and (/sup 32/P) P/sub i/ into phosphatidylinositol of hamster brown fat cells in a concentration-dependent manner. Both responses were preferentially inhibited by prazosin as compared with yohimbine, indicating alpha 1 specificity. Uniquely, prazosin inhibition of phenylephrine-stimulated phosphatidylinositol metabolism had two components, since 30% of the response was inhibited by less than 1 nM prazosin, 10 nM gave no further inhibition, and 100 nM prazosin completely inhibited the response. The phosphatidylinositol response was still present in Ca/sup 2/+-free buffer, although reduced in magnitude. The concentration relationships of the effects of agonists and antagonists were compared with those of previous results of (/sup 3/H)prazosin binding and with phenylephrine potency to compete for binding. On the basis of these comparisons, it is suggested that the highly prazosin-sensitive part of the phosphatidylinositol response may be closely associated with receptor occupation.

  10. Utility of thymosin alpha-1 (Zadaxin) as a co-adjuvant in influenza vaccines: a review.

    PubMed

    Panatto, D; Amicizia, D; Lai, P L; Camerini, R; De Rosa, A; Gasparini, R

    2011-09-01

    Influenza constitutes a serious problem for healthcare and social services worldwide, owing to its pattern and the severity of its complications in some categories of subjects at risk, such as the elderly and immunocompromised individuals. The only really effective means of combating influenza is vaccination. The elderly and immunocompromised subjects are refractory or low responders to vaccination. The need for ever more immunogenic and efficacious influenza vaccines, especially for subjects at risk, has prompted the development of adjuvated vaccines. With a view to enhancing the immune response in the elderly and in subjects at risk, the possibility of co-administering immunostimulants as Thymosin alpha-1 (Talpha1) with influenza vaccines has been investigated. Talpha1 is a biologically active peptide made up of 28 amino acids that can enhance T-cells, dendritic cell and antibody responses, modulate cytokines and chemokines production. Several studies were conducted and showed that Talpha1 ameliorate the performanc of influenza vaccination in elderly and subjects at risk. Although further studies on co-adjuvants are necessary, the future prospects of producing ever more efficacious influenza vaccines appear very promising.

  11. Mutation in collagen II alpha 1 isoforms delineates Stickler and Wagner syndrome phenotypes

    PubMed Central

    Tran-Viet, Khanh-Nhat; Soler, Vincent; Quiette, Valencia; Powell, Caldwell; Yanovitch, Tammy; Metlapally, Ravikanth; Luo, Xiaoyan; Katsanis, Nicholas; Nading, Erica

    2013-01-01

    Purpose Stickler syndrome is an arthro-ophthalmopathy with phenotypic overlap with Wagner syndrome. The common Stickler syndrome type I is inherited as an autosomal dominant trait, with causal mutations in collagen type II alpha 1 (COL2A1). Wagner syndrome is associated with mutations in versican (VCAN), which encodes for a chondroitin sulfate proteoglycan. A three-generation Caucasian family variably diagnosed with either syndrome was screened for sequence variants in the COL2A1 and VCAN genes. Methods Genomic DNA samples derived from saliva were collected from all family members (six affected and four unaffected individuals). Complete sequencing of COL2A1 and VCAN was performed on two affected individuals. Direct sequencing of remaining family members was conducted if the discovered variants followed segregation. Results A base-pair substitution (c.258C>A) in exon 2 of COL2A1 cosegregated with familial disease status. This known mutation occurs in a highly conserved site that causes a premature stop codon (p.C86X). The mutation was not seen in 1,142 ethnically matched control DNA samples. Conclusions Premature stop codons in COL2A1 exon 2 lead to a Stickler syndrome type I ocular-only phenotype with few or no systemic manifestations. Mutation screening of COL2A1 exon 2 in families with autosomal dominant vitreoretinopathy is important for accurate clinical diagnosis. PMID:23592912

  12. Alpha-1 adrenergic receptors gate rapid orientation-specific reduction in visual discrimination.

    PubMed

    Treviño, Mario; Frey, Sebastian; Köhr, Georg

    2012-11-01

    Prolonged imbalance in sensory experience leads to dramatic readjustments in cortical representation. Neuromodulatory systems play a critical role in habilitating experience-induced plasticity and regulate memory processes in vivo. Here, we show that a brief period of intense patterned visual stimulation combined with systemic activation of alpha-1 adrenergic neuromodulator receptors (α(1)-ARs) leads to a rapid, reversible, and NMDAR-dependent depression of AMPAR-mediated transmission from ascending inputs to layer II/III pyramidal cells in the visual cortex of young and adult mice. The magnitude of this form of α(1)-AR long-term depression (LTD), measured ex vivo with miniature EPSC recordings, is graded by the number of orientations used during visual experience. Moreover, behavioral tests of visual function following the induction of α(1)-AR LTD reveal that discrimination accuracy of sinusoidal drifting gratings is selectively reduced at high spatial frequencies in a reversible, orientation-specific, and NMDAR-dependent manner. Thus, α(1)-ARs enable rapid cortical synaptic depression which correlates with an orientation-specific decrease in visual discrimination. These findings contribute to our understanding of how adrenergic receptors interact with neuronal networks in response to changes in active sensory experience to produce adaptive behavior.

  13. Cerebral artery alpha-1 AR subtypes: high altitude long-term acclimatization responses.

    PubMed

    Goyal, Ravi; Goyal, Dipali; Chu, Nina; Van Wickle, Jonathan; Longo, Lawrence D

    2014-01-01

    In response to hypoxia and other stress, the sympathetic (adrenergic) nervous system regulates arterial contractility and blood flow, partly through differential activities of the alpha1 (α1) - adrenergic receptor (AR) subtypes (α1A-, α1B-, and α1D-AR). Thus, we tested the hypothesis that with acclimatization to long-term hypoxia (LTH), contractility of middle cerebral arteries (MCA) is regulated by changes in expression and activation of the specific α1-AR subtypes. We conducted experiments in MCA from adult normoxic sheep maintained near sea level (300 m) and those exposed to LTH (110 days at 3801 m). Following acclimatization to LTH, ovine MCA showed a 20% reduction (n = 5; P<0.05) in the maximum tension achieved by 10-5 M phenylephrine (PHE). LTH-acclimatized cerebral arteries also demonstrated a statistically significant (P<0.05) inhibition of PHE-induced contractility in the presence of specific α1-AR subtype antagonists. Importantly, compared to normoxic vessels, there was significantly greater (P<0.05) α1B-AR subtype mRNA and protein levels in LTH acclimatized MCA. Also, our results demonstrate that extracellular regulated kinase 1 and 2 (ERK1/2)-mediated negative feedback regulation of PHE-induced contractility is modulated by α1B-AR subtype. Overall, in ovine MCA, LTH produces profound effects on α1-AR subtype expression and function.

  14. Thyroid hormone receptor alpha1 follows a cooperative CRM1/calreticulin-mediated nuclear export pathway.

    PubMed

    Grespin, Matthew E; Bonamy, Ghislain M C; Roggero, Vincent R; Cameron, Nicole G; Adam, Lindsay E; Atchison, Andrew P; Fratto, Victoria M; Allison, Lizabeth A

    2008-09-12

    The thyroid hormone receptor alpha1 (TRalpha) exhibits a dual role as an activator or repressor of its target genes in response to thyroid hormone (T(3)). Previously, we have shown that TRalpha, formerly thought to reside solely in the nucleus bound to DNA, actually shuttles rapidly between the nucleus and cytoplasm. An important aspect of the shuttling activity of TRalpha is its ability to exit the nucleus through the nuclear pore complex. TRalpha export is not sensitive to treatment with the CRM1-specific inhibitor leptomycin B (LMB) in heterokaryon assays, suggesting a role for an export receptor other than CRM1. Here, we have used a combined approach of in vivo fluorescence recovery after photobleaching experiments, in vitro permeabilized cell nuclear export assays, and glutathione S-transferase pull-down assays to investigate the export pathway used by TRalpha. We show that, in addition to shuttling in heterokaryons, TRalpha shuttles rapidly in an unfused monokaryon system as well. Furthermore, our data show that TRalpha directly interacts with calreticulin, and point to the intriguing possibility that TRalpha follows a cooperative export pathway in which both calreticulin and CRM1 play a role in facilitating efficient translocation of TRalpha from the nucleus to cytoplasm. PMID:18641393

  15. Collagen Type XI Alpha 1 Expression in Intraductal Papillomas Predicts Malignant Recurrence.

    PubMed

    Freire, Javier; García-Berbel, Lucia; García-Berbel, Pilar; Pereda, Saray; Azueta, Ainara; García-Arranz, Pilar; De Juan, Ana; Vega, Alfonso; Hens, Ángela; Enguita, Ana; Muñoz-Cacho, Pedro; Gómez-Román, Javier

    2015-01-01

    Despite the progress achieved in the treatment of breast cancer, there are still many unsolved clinical issues, being the diagnosis, prognosis, and treatment of papillary diseases, one of the highest challenges. Because of its unpredictable clinical behavior, treatment of intraductal papilloma has generated a great controversy. Even though considered as a benign lesion, it presents high rate of malignant recurrence. This is the reason why there are clinicians supporting a complete excision of the lesion, while others support an only expectant follow-up. Previous results of our group suggested that procollagen 11 alpha 1 (pro-COL11A1) expression correlates with infiltrating phenotype in breast lesions. We analyzed the correlation between expression of pro-COL11A1 in intraductal papilloma and their risk of malignant recurrence. Immunohistochemistry of pro-COL11A1 was performed in 62 samples of intraductal papilloma. Ten out 11 cases relapsed as carcinoma presents positive staining for COL11A1, while just 17 out of 51 cases with benign behaviour present immunostaining. There were significant differences (P < 0.0001) when comparing patients with malignant recurrence versus nonmalignant relapse patients. These data suggest that pro-COL11A1 expression is a highly sensitive biomarker to predict malignant relapse of intraductal papilloma and it can be used as indicative factor for prevention programs.

  16. Alpha (1,2)-fucosyltransferase M307A polymorphism improves piglet survival.

    PubMed

    Kim, Kyungtae; Nguyen, Dinh Truong; Choi, Minkyung; Kim, Jin-Hoi; Seo, Han Geuk; Dadi, Hailu; Cha, Se-Yeoun; Seo, Kunho; Lee, Yun-Mi; Kim, Jong-Joo; Park, Chankyu

    2013-01-01

    To confirm the beneficial effects of alpha (1,2)-fucosyltransferase (FUT1) M307 (A) on piglet survival on commercial farms, we performed PCR-RFLP analysis of FUT1 M307 in successfully marketed (n = 245) and disease affected/deceased pigs during weaning (n = 252) at a commercial farm. We also evaluated the FUT1 genotypes of 190 healthy pigs from three different genetic backgrounds. The distribution of genotypes differed between the successfully marketed and disease affected/deceased pig groups. The frequency of the A allele, associated with resistance to edema and post-weaning diarrhea, was higher in the post-weaning survival group (0.21) than in the non-survival group (0.16, P < 0.05). The odds ratio for piglet survival between AA and GG genotypes was 1.98; thus, piglet survival for individuals with the AA genotype was almost two-fold greater than for GG individuals. The FUT1 gene polymorphism can be used as an effective marker for selection programs to improve post-weaning piglet survival.

  17. An amino acid substitution (Gly853-->Glu) in the collagen alpha 1(II) chain produces hypochondrogenesis.

    PubMed

    Bogaert, R; Tiller, G E; Weis, M A; Gruber, H E; Rimoin, D L; Cohn, D H; Eyre, D R

    1992-11-01

    The spondyloepiphyseal dysplasia subclassification of bone dysplasias includes achondrogenesis, hypochondrogenesis, and spondyloepiphyseal dysplasia congenita. The phenotypic expression of these disorders ranges from mild to perinatal lethal forms. We report the detection and partial characterization of a defect in type II collagen in a perinatal lethal form of hypochondrogenesis. Electrophoresis in sodium dodecyl sulfate-polyacrylamide of CB peptides (where CB represents cyanogen bromide) from type II collagen of the diseased cartilage showed a doublet band for peptide alpha 1(II)CB10 and evidence for post-translational overmodification of the major peptides (CB8, CB10, and CB11) seen as a retarded electrophoretic mobility. Peptide CB10 was digested by endoproteinase Asp-N; and on reverse-phase high pressure liquid chromatography, fragments of abnormal mobility were noted. Sequence analysis of a unique peptide D12 revealed a single amino acid substitution (Gly-->Glu) at position 853 of the triple helical domain. This was confirmed by sequence analysis of amplified COL2A1 cDNA, which revealed a single nucleotide substitution (GGA-->GAA) in 5 of 10 clones. Electron micrographs of the diseased cartilage showed a sparse extracellular matrix and chondrocytes containing dilated rough endoplasmic reticulum, which suggested impaired assembly and secretion of the mutant protein. This case further documents the molecular basis of the spondyloepiphyseal dysplasia spectrum of chondrodysplasias as mutations in COL2A1. PMID:1429602

  18. Collagen Type XI Alpha 1 Expression in Intraductal Papillomas Predicts Malignant Recurrence

    PubMed Central

    Freire, Javier; García-Berbel, Lucia; García-Berbel, Pilar; Pereda, Saray; Azueta, Ainara; García-Arranz, Pilar; De Juan, Ana; Vega, Alfonso; Hens, Ángela; Enguita, Ana; Muñoz-Cacho, Pedro; Gómez-Román, Javier

    2015-01-01

    Despite the progress achieved in the treatment of breast cancer, there are still many unsolved clinical issues, being the diagnosis, prognosis, and treatment of papillary diseases, one of the highest challenges. Because of its unpredictable clinical behavior, treatment of intraductal papilloma has generated a great controversy. Even though considered as a benign lesion, it presents high rate of malignant recurrence. This is the reason why there are clinicians supporting a complete excision of the lesion, while others support an only expectant follow-up. Previous results of our group suggested that procollagen 11 alpha 1 (pro-COL11A1) expression correlates with infiltrating phenotype in breast lesions. We analyzed the correlation between expression of pro-COL11A1 in intraductal papilloma and their risk of malignant recurrence. Immunohistochemistry of pro-COL11A1 was performed in 62 samples of intraductal papilloma. Ten out 11 cases relapsed as carcinoma presents positive staining for COL11A1, while just 17 out of 51 cases with benign behaviour present immunostaining. There were significant differences (P < 0.0001) when comparing patients with malignant recurrence versus nonmalignant relapse patients. These data suggest that pro-COL11A1 expression is a highly sensitive biomarker to predict malignant relapse of intraductal papilloma and it can be used as indicative factor for prevention programs. PMID:26448946

  19. Alpha-1, alpha-2, and beta adrenergic signal transduction in cultured uterine myocytes.

    PubMed

    Phillippe, M; Saunders, T; Bangalore, S

    1990-04-01

    The following studies were undertaken to develop a cultured uterine myocyte model which would allow further clarification of the adrenergic signal transduction mechanisms utilized by these myocytes. After mechanical removal of the endometrium, rabbit uterine myocytes were isolated by an overnight enzymatic disaggregation using collagenase and DNase I. The isolated myocytes were maintained in culture in 75-cm2 flasks containing Waymouth's MB 751/1 medium-10% fetal bovine serum along with 10(-8) M estradiol, penicillin, streptomycin, and Fungizone. The phase contrast and electron micrographic appearance of these cells was consistent with that previously reported for smooth muscle myocytes in culture. Immunocytochemical studies utilizing monoclonal anti-alpha-smooth muscle actin antibodies confirmed the presence of smooth muscle actin in these cultured myocytes. Western blot studies similarly confirmed the presence of alpha-smooth muscle actin in rabbit myometrial tissue and the cultured myocytes, both the primary and F1 generation. After prelabeling the myocytes with [3H]inositol, adrenergic stimulation experiments demonstrated alpha-1 receptor mediated stimulation of inositol phosphates. Beta receptor stimulation experiments confirmed cAMP production in these cultured myocytes, and the ability of clonidine, an alpha-2 agonist, to inhibit forskolin stimulated cAMP production confirmed the presence of functional alpha-2 adrenergic receptors in these myocytes. In conclusion, these cultured rabbit uterine myocytes have provided an in vitro model which can be utilized to further clarify the adrenergic receptor signal transduction mechanisms in genital tract smooth muscle.

  20. Selected metabolic aspects of elastin and collagen fiber proteolysis in diseases of the respiratory system – the significance of α1 antitrypsin deficiency

    PubMed Central

    Rokicki, Wojciech; Karuś, Krzysztof

    2016-01-01

    The process of elastin and collagen fiber destruction was presented based on the example of the changes taking place in the course of chronic obstructive pulmonary disease and primary spontaneous pneumothorax. In 1963, when analyzing patients with α1 antitrypsin deficiency, Dr Laurell and Dr Eriksson hypothesized that elastolysis plays a role in the pathogenesis of emphysema, which marked the beginning of studies aimed at analyzing this process. The present work concerns new scientific reports regarding the hypothesis. The most important risk factors include protease-antiprotease imbalance and α1 antitrypsin protein deficiency. PMID:27785139

  1. Compartmentation of alpha 1 and alpha 2 GABA(A) receptor subunits within rat extended amygdala: implications for benzodiazepine action.

    PubMed

    Kaufmann, Walter A; Humpel, Christian; Alheid, George F; Marksteiner, Josef

    2003-02-21

    The extended amygdala, a morphological and functional entity within the basal forebrain, is a neuronal substrate for emotional states like fear and anxiety. Anxiety disorders are commonly treated by benzodiazepines that mediate their action via GABA(A) receptors. The binding properties and action of benzodiazepines depend on the alpha-subunit profile of the hetero-pentameric receptors: whereas the alpha1 subunit is associated with benzodiazepine type I pharmacology and reportedly mediates sedative as well as amnesic actions of benzodiazepines, the alpha2 subunit confers benzodiazepine type II pharmacology and mediates the anxiolytic actions of benzodiazepines. We determined the localization of alpha1 and alpha2 subunits within the extended amygdala, identified by secretoneurin immunostaining, to define the morphological substrates for the diverse benzodiazepine actions. A moderate expression of the alpha1 subunit could be detected in compartments of the medial subdivision and a strong expression of the alpha2 subunit throughout the central subdivision. It is concluded that the alpha1 and alpha2 subunits are differentially expressed within the extended amygdala, indicating that this structure is compartmentalized with respect to function and benzodiazepine action. PMID:12573516

  2. Isolation of a putative receptor from Zea mays microsomal membranes that interacts with the G-protein, GP alpha 1.

    PubMed

    Wise, A; Thomas, P G; White, I R; Millner, P A

    1994-12-19

    The C-terminal region of a heterotrimeric G-protein alpha-subunit is known to be one of the principal determinants governing its interaction with its cognate receptor. Use of an oligopeptide corresponding to the fifteen C-terminal residues of the Arabidopsis G alpha-subunit (GP alpha 1), as an affinity ligand, led to the resolution of a tightly binding 37 kDa membrane polypeptide from detergent solubilised Zea microsomal fraction membranes. An identical polypeptide bound tightly to an affinity matrix containing recombinant GP alpha 1 protein as ligand: binding and release of this 37 kDa protein was dependent on the activation state of GP alpha 1 which was regulated by inclusion or omission of the G-protein activator AlF-4. Finally, the isolated 37 kDa protein was labelled with the lectin concanavalin A, indicating it to be glycosylated. These data are consistent with the identity of the 37 kDa membrane polypeptide as a receptor that interacts with the Zea homologue of GP alpha 1. PMID:7805845

  3. Transcription factors nuclear factor I and Sp1 interact with the murine collagen alpha 1 (I) promoter.

    PubMed Central

    Nehls, M C; Rippe, R A; Veloz, L; Brenner, D A

    1991-01-01

    The collagen alpha 1(I) promoter, which is efficiently transcribed in NIH 3T3 fibroblasts, contains four binding sites for trans-acting factors, as demonstrated by DNase I protection assays (D. A. Brenner, R. A. Rippe, and L. Veloz, Nucleic Acids Res. 17:6055-6064, 1989). This study characterizes the DNA-binding proteins that interact with the two proximal footprinted regions, both of which contain a reverse CCAAT box and a G + C-rich 12-bp direct repeat. Analysis by DNase I protection assays, mobility shift assays, competition with specific oligonucleotides, binding with recombinant proteins, and reactions with specific antisera showed that the transcriptional factors nuclear factor I (NF-I) and Sp1 bind to these two footprinted regions. Because of overlapping binding sites, NF-I binding and Sp1 binding appear to be mutually exclusive. Overexpression of NF-I in cotransfection experiments with the alpha 1(I) promoter in NIH 3T3 fibroblasts increased alpha 1(I) expression, while Sp1 overexpression reduced this effect, as well as basal promoter activity. The herpes simplex virus thymidine kinase promoter, which contains independent NF-I- and Sp1-binding sites, was stimulated by both factors. Therefore, expression of the collagen alpha 1(I) gene may depend on the relative activities of NF-I and Sp1. Images PMID:2072909

  4. Identification of alpha-1 acid glycoprotein (AGP) as a potential marker of impaired growth in the newborn piglet

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two studies were conducted to investigate the relationship between the circulating levels of the acute phase proteins haptoglobin (HP) and alpha 1 acid glycoprotein (AGP) and growth potential in neonatal pigs. In runts, the circulating level of AGP, but not HP in serum of newborn piglets was higher...

  5. Carbohydrate binding properties of banana (Musa acuminata) lectin I. Novel recognition of internal alpha1,3-linked glucosyl residues.

    PubMed

    Mo, H; Winter, H C; Van Damme, E J; Peumans, W J; Misaki, A; Goldstein, I J

    2001-05-01

    Examination of lectins of banana (Musa acuminata) and the closely related plantain (Musa spp.) by the techniques of quantitative precipitation, hapten inhibition of precipitation, and isothermal titration calorimetry showed that they are mannose/glucose binding proteins with a preference for the alpha-anomeric form of these sugars. Both generate precipitin curves with branched chain alpha-mannans (yeast mannans) and alpha-glucans (glycogens, dextrans, and starches), but not with linear alpha-glucans containing only alpha1,4- and alpha1,6-glucosidic bonds (isolichenan and pullulan). The novel observation was made that banana and plantain lectins recognize internal alpha1,3-linked glucosyl residues, which occur in the linear polysaccharides elsinan and nigeran. Concanavalin A and lectins from pea and lentil, also mannose/glucose binding lectins, did not precipitate with any of these linear alpha-glucans. This is, the authors believe, the first report of the recognition of internal alpha1,3-glucosidic bonds by a plant lectin. It is possible that these lectins are present in the pulp of their respective fruit, complexed with starch.

  6. Enhanced Noradrenergic Activity Potentiates Fear Memory Consolidation and Reconsolidation by Differentially Recruiting alpha1- and beta-Adrenergic Receptors

    ERIC Educational Resources Information Center

    Gazarini, Lucas; Stern, Cristina A. Jark; Carobrez, Antonio P.; Bertoglio, Leandro J.

    2013-01-01

    Consolidation and reconsolidation are phases of memory stabilization that diverge slightly. Noradrenaline is known to influence both processes, but the relative contribution of alpha1- and beta-adrenoceptors is unclear. The present study sought to investigate this matter by comparing their recruitment to consolidate and/or reconsolidate a…

  7. Rare deficiency {alpha}{sub 1} Antitrypsin variants; current status and SSCP analysis

    SciTech Connect

    Billingsley, G.D.; Cox, D.W.

    1994-09-01

    The serine protease inhibitor {alpha}{sub 1} Antitrypsin ({alpha}{sub 1}AT) is an inhibitor of neutrophil elastase. A deficiency of {alpha}{sub 1}AT (< 20% of the normal amount of {alpha}{sub 1}AT) is associated with early-onset emphysema and childhood liver disease. The most common deficiency allele, PI{sup *}Z, has a frequency of 1-2% in the North American white population. Several rare deficiency alleles (including null (QO) alleles; < 1% of normal) with a combined frequency of approximately 10{sup -4}, have been reported. Of 24 sequenced deficiency variants, the defect in 15 has been proven to be due to gene deletion (2), mRNA degradation (2), error in mRNA processing (1), intracellular protein accumulation (5) and intracellular protein degradation (5). We have determined conditions for detection of new mutations. We have screened DNA from 20 individuals carrying rare deficiency alleles. In some individuals, RFLP haplotype analysis suggested the presence of a new variant. The root alleles, M1 (Ala 213) or M1 (Val 213), and the presence of variants whose mutant sequence alters a restriction endonuclease site were determined by digestion of the amplified exon. Mutation detection was performed by SSCP analysis of each of the four coding exons followed by direct sequencing of the amplified exon. 12 of 14 known mutations (85%) were detected by SSCP analysis. We detected a new null allele in a patient that also carries the QO{sup *}hongkong allele. C to A transversion at the third nucleotide of codon 38 creates a stop codon on the M1(Val 213) root allele. This new variant allele has been named PI{sup *}QOkowloon. Characterization of the mutations leading to {alpha}{sub 1}AT deficiency allows delineation of amino acids critical for stability, for normal secretion and for normal function.

  8. α1-Antitrypsin reduces rhinovirus infection in primary human airway epithelial cells exposed to cigarette smoke

    PubMed Central

    Berman, Reena; Jiang, Di; Wu, Qun; Chu, Hong Wei

    2016-01-01

    Human rhinovirus (HRV) infections target airway epithelium and are the leading cause of acute exacerbations of COPD. Cigarette smoke (CS) increases the severity of viral infections, but there is no effective therapy for HRV infection. We determined whether α1-antitrypsin (A1AT) reduces HRV-16 infection in CS-exposed primary human airway epithelial cells. Brushed bronchial epithelial cells from normal subjects and patients diagnosed with COPD were cultured at air–liquid interface to induce mucociliary differentiation. These cells were treated with A1AT or bovine serum albumin for 2 hours and then exposed to air or whole cigarette smoke (WCS) with or without HRV-16 (5×104 50% Tissue Culture Infective Dose [TCID50]/transwell) infection for 24 hours. WCS exposure significantly increased viral load by an average of fivefold and decreased the expression of antiviral genes interferon-λ1, OAS1, and MX1. When A1AT was added to WCS-exposed cells, viral load significantly decreased by an average of 29-fold. HRV-16 infection significantly increased HRV-16 receptor intercellular adhesion molecule-1 messenger RNA expression in air-exposed cells, which was decreased by A1AT. A1AT-mediated reduction of viral load was not accompanied by increased epithelial antiviral gene expression or by inhibiting the activity of 3C protease involved in viral replication or maturation. Our findings demonstrate that A1AT treatment prevents a WCS-induced increase in viral load and for the first time suggest a therapeutic effect of A1AT on HRV infection. PMID:27354786

  9. Z α1-Antitrypsin Confers a Proinflammatory Phenotype That Contributes to Chronic Obstructive Pulmonary Disease

    PubMed Central

    Alam, Samuel; Li, Zhenjun; Atkinson, Carl; Jonigk, Danny; Janciauskiene, Sabina

    2014-01-01

    Rationale: Severe α1-antitrypsin deficiency caused by the Z variant (Glu342Lys; ZZ-AT) is a well-known genetic cause for emphysema. Although severe lack of antiproteinase protection is the critical etiologic factor for ZZ-AT–associated chronic obstructive pulmonary disease (COPD), some reports have suggested enhanced lung inflammation as a factor in ZZ-AT homozygotes. Objectives: To provide molecular characterization of inflammation in ZZ-AT. Methods: Inflammatory cell and cytokine profile (nuclear factor-κB, IL-6, tumor necrosis factor-α), intracellular polymerization of Z-AT, and endoplasmic reticulum (ER) stress markers (protein kinase RNA–like ER kinase, activator transcription factor 4) were assessed in transgenic mice and transfected cells in response to cigarette smoke, and in explanted lungs from ZZ and MM individuals with severe COPD. Measurements and Main Results: Compared with M-AT, transgenic Z-AT mice lungs exposed to cigarette smoke had higher levels of pulmonary cytokines, neutrophils, and macrophages and an exaggerated ER stress. Similarly, the ER overload response was greater in lungs from ZZ-AT homozygotes with COPD, and was particularly found in pulmonary epithelial cells. Cigarette smoke increased intracellular Z-AT polymers, ER overload response, and proinflammatory cytokine release in Z-AT–expressing pulmonary epithelial cells, which could be prevented with an inhibitor of polymerization, an antioxidant, and an inhibitor of protein kinase RNA–like ER kinase. Conclusions: We show here that aggregation of intracellular mutant Z-AT invokes a specific deleterious cellular inflammatory phenotype in COPD. Oxidant-induced intracellular polymerization of Z-AT in epithelial cells causes ER stress, and promotes excess cytokine and cellular inflammation. This pathway is likely to contribute to the development of COPD in ZZ-AT homozygotes, and therefore merits further investigation. PMID:24592811

  10. Chronic obstructive pulmonary disease in α1-antitrypsin PI MZ heterozygotes: a meta-analysis

    PubMed Central

    Hersh, C; Dahl, M; Ly, N; Berkey, C; Nordestgaard, B; Silverman, E

    2004-01-01

    Background: Severe α1-antitrypsin deficiency, usually related to homozygosity for the protease inhibitor (PI) Z allele, is a proven genetic risk factor for chronic obstructive pulmonary disease (COPD). The risk of COPD in PI MZ heterozygous individuals is controversial. Methods: A search of MEDLINE from January 1966 to May 2003 identified studies that examined the risk of COPD in PI MZ individuals and studies that measured forced expiratory volume in 1 second (FEV1) in heterozygotes. Results: In 16 studies that reported COPD as a categorical outcome, the combined odds ratio (OR) for PI MZ versus PI MM (normal genotype) was 2.31 (95% CI 1.60 to 3.35). The summary OR was higher in case-control studies (OR 2.97; 95% CI 2.08 to 4.26) than in cross sectional studies (OR 1.50; 95% CI 0.97 to 2.31) and was attenuated in studies that adjusted for cigarette smoking (OR 1.61; 95% CI 0.92 to 2.81). In seven studies that reported FEV1 as a continuous outcome there was no difference in mean FEV1 between PI MM and PI MZ individuals. Conclusions: Case-control studies showed increased odds of COPD in PI MZ individuals, but this finding was not confirmed in cross sectional studies. Variability in study design and quality limits the interpretation. These results are consistent with a small increase in risk of COPD in all PI MZ individuals or a larger risk in a subset. Future studies that adjust for smoking and include other COPD related phenotypes are required to conclusively determine the risk of COPD in PI MZ heterozygotes. PMID:15454649

  11. Effects of a caffeine-free Cola nitida nuts extract on elastase/alpha-1-proteinase inhibitor balance.

    PubMed

    Daels-Rakotoarison, Dominique A; Kouakou, Gisèle; Gressier, Bernard; Dine, Thierry; Brunet, Claude; Luyckx, Michel; Bailleul, François; Trotin, Francis

    2003-11-01

    In an infection, polymorphonuclear neutrophils (PMN) become activated and they produce oxidizing compounds and elastase in the extracellular medium. Alpha-1-proteinase inhibitor (alpha1PI), a protease inhibitor which is inactivated by oxidants, is the main endogenous inhibitor of elastase helping to limit excessive elastase activity. This study evaluates the ability of a plant extract, Cola nitida nuts, to protect alpha1PI from inactivation by oxidizing compounds as reactive oxygen species. On the one hand, we have evaluated the direct effect of cola nut extract on neutrophil elastase, and on the H(2)O(2) and myeloperoxidase (MPO)-H(2)O(2) system via cell-free systems. Results showed that cola nut extract scavenges H(2)O(2) and therefore protects alpha1PI from HOCl which is produced from the MPO-H(2)O(2) system. Experiments also showed that cola extract has the capacity to limit elastase activity. On the other hand, we have worked on cellular systems including isolated PMN with the aim to study the effect of cola extract on PMN metabolism. PMN were stimulated with PMA, calcium ionophore or fMLP. Each stimulant possesses its own stimulation pathway. According to the inhibitory concentration obtained at 50%, the results on cellular systems led to the conclusion that cola extract can reduce elastase liberation from PMN. It can then be concluded that cola nut extract can protect alpha1PI from inactivation, and has an effect both on elastase liberation and elastase activity. The cola nut extract effect is rather biased towards a reduction in elastase release, thus limiting the injurious effects caused by this enzyme.

  12. Transient expression of laminin {alpha}1 chain in regenerating murine liver: Restricted localization of laminin chains and nidogen-1

    SciTech Connect

    Kikkawa, Yamato . E-mail: yamato@sapmed.ac.jp; Mochizuki, Yoichi; Miner, Jeffrey H.; Mitaka, Toshihiro

    2005-04-15

    Most interstitia between epithelial and endothelial cells contain basal laminae (BLs), as defined by electron microscopy. However, in liver, the sinusoidal interstitium (called space of Disse) between hepatocytes and sinusoidal endothelial cells (SECs) lacks BLs. Because laminins are major components of BLs throughout the body, whether laminins exist in sinusoids has been a controversial issue. Despite recent advances, the distribution and expression of laminin chains have not been well defined in mammalian liver. Here, using a panel of antibodies, we examined laminins in normal and regenerating mouse livers. Of {alpha} chains, {alpha}5 was widely observed in all BLs except for sinusoids, while the other {alpha} chains were variously expressed in Glisson's sheath and central veins. Laminin {gamma}1 was also distributed to all BLs except for sinusoids. Although the {beta}2 chain was observed in all BLs and sinusoids, the expression of {beta}1 chain was restricted to Glisson's sheath. Detailed analysis of regenerating liver revealed that {alpha}1 and {gamma}1 chains appeared in sinusoids and were produced by stellate cells. The staining of {alpha}1 and {gamma}1 chains reached its maximum intensity at 6 days after two-thirds partial hepatectomy (PHx). Moreover, in vitro studies showed that {alpha}1-containing laminin promoted spreading of sinusoidal endothelial cells (SECs) isolated from normal liver, but not other hepatic cells. In addition, SECs isolated from regenerating liver elongated pseudopodia on {alpha}1-containing laminin more so than did cells from normal liver. The transient expression of laminin {alpha}1 may promote formation of sinusoids after PHx.

  13. Stress-induced decrease of uterine blood flow in sheep is mediated by alpha 1-adrenergic receptors.

    PubMed

    Dreiling, Michelle; Bischoff, Sabine; Schiffner, Rene; Rupprecht, Sven; Kiehntopf, Michael; Schubert, Harald; Witte, Otto W; Nathanielsz, Peter W; Schwab, Matthias; Rakers, Florian

    2016-09-01

    Prenatal maternal stress can be transferred to the fetus via a catecholamine-dependent decrease of uterine blood flow (UBF). However, it is unclear which group of adrenergic receptors mediates this mechanism of maternal-fetal stress transfer. We hypothesized that in sheep, alpha 1-adrenergic receptors may play a key role in catecholamine mediated UBF decrease, as these receptors are mainly involved in peripheral vasoconstriction and are present in significant number in the uterine vasculature. After chronic instrumentation at 125 ± 1 days of gestation (dGA; term 150 dGA), nine pregnant sheep were exposed at 130 ± 1 dGA to acute isolation stress for one hour without visual, tactile, or auditory contact with their flockmates. UBF, blood pressure (BP), heart rate (HR), stress hormones, and blood gases were determined before and during this isolation challenge. Twenty-four hours later, experiments were repeated during alpha 1-adrenergic receptor blockage induced by a continuous intravenous infusion of urapidil. In both experiments, ewes reacted to isolation with an increase in serum norepinephrine, cortisol, BP, and HR as typical signs of activation of sympatho-adrenal and the hypothalamic-pituitary-adrenal axis. Stress-induced UBF decrease was prevented by alpha 1-adrenergic receptor blockage. We conclude that UBF decrease induced by maternal stress in sheep is mediated by alpha 1-adrenergic receptors. Future studies investigating prevention strategies of impact of prenatal maternal stress on fetal health should consider selective blockage of alpha 1-receptors to interrupt maternal-fetal stress transfer mediated by utero-placental malperfusion.

  14. The GABA(A) receptor alpha1 subtype in the ventral pallidum regulates alcohol-seeking behaviors.

    PubMed

    Harvey, Scott C; Foster, Katrina L; McKay, Pete F; Carroll, Michelle R; Seyoum, Regat; Woods, James E; Grey, Collette; Jones, Cecily M; McCane, Shannan; Cummings, Rancia; Mason, Dynesha; Ma, Chunrong; Cook, James M; June, Harry L

    2002-05-01

    We investigated the potential role of the alpha1-containing GABA(A) receptor in regulating the reinforcing properties of alcohol. To accomplish this, we developed 3-propoxy-beta-carboline hydrochloride (3-PBC), a mixed agonist-antagonist benzodiazepine site ligand with binding selectivity at the alpha1 receptor. We then tested the capacity of 3-PBC to block alcohol-maintained responding in the ventral pallidum (VP), a novel alcohol reward substrate, which primarily expresses the alpha1-receptor isoform. Our results demonstrated that bilateral microinfusion of 3-PBC (0.5-40 microg) in the anterior and medial VP produced marked reductions in alcohol-maintained responding in a genetically selected rodent model of alcohol drinking. The VP infusions showed both neuroanatomical and reinforcer specificity because no effects were seen in sites dorsal to the VP (e.g., nucleus accumbens, caudate putamen). The saccharin-maintained responding was reduced only with the highest dose (40 microg). Parenteral injections of 3-PBC (1-20 mg/kg) also showed a similar selectivity on alcohol-maintained responding. Complementary in vitro studies revealed that 3-PBC exhibited a low partial agonist efficacy profile at recombinant diazepam-sensitive receptors (e.g., alpha1beta3gamma2, alpha2beta3gamma, and alpha3beta3gamma2). The selective suppression of 3-PBC on alcohol-maintained responding after central and parenteral administrations, together with its low-efficacy agonist profile, suggest that the reduction in alcohol-maintained behaviors was not attributable to a general suppression on consummatory behaviors. These results demonstrate that the alpha1-containing GABA(A) receptors in both the anterior and medial VP are important in regulating the reinforcing properties of alcohol. These receptors represent novel targets in the design and development of pharmacotherapies for alcohol-dependent subjects. PMID:11978852

  15. Immunogenic Gal alpha 1----3Gal carbohydrate epitopes are present on pathogenic American Trypanosoma and Leishmania.

    PubMed

    Avila, J L; Rojas, M; Galili, U

    1989-04-15

    Anti-Gal is a natural antibody present in unusually high concentrations in human sera. It constitutes as much as 1% of circulating IgG and displays a distinct specificity for the Gal alpha 1----3Gal carbohydrate epitope. In the present study, we have found in the sera of patients with Chagas' disease and Leishmania infection anti-Gal titers 10- and 16-fold higher than that of healthy or bacteria-infected individuals. This increase in anti-Gal titer seemed to be the result of a specific immune response toward parasitic Gal alpha 1----3Gal epitopes. Binding studies of affinity chromatography-purified anti-Gal antibodies to Trypanosoma cruzi and American Leishmania parasites indeed demonstrated the presence of Gal alpha 1----3Gal epitopes on these parasites. This finding was supported by the observed binding to the parasites of two additional Gal alpha 1----3Gal recognizing molecules: the mAb Gal-13, and the lectin, Bandeiraea simplicifolia I B4. Furthermore, the binding of both anti-Gal antibody and of the B. simplicifolia I B4 lectin could be inhibited by galactose, and not glucose. In addition, removal of the terminal alpha-galactosyl residues from the parasites by pretreatment with alpha-galactosidase, or the oxidation of the binding epitopes by periodate prevented the subsequent binding of both the antibody and the lectin. A crude leishmanial lipid extract readily bound these three reagents, suggesting that at least part of these epitopes are of a glycolipid nature. These Gal alpha 1----3Gal epitopes may thus serve as an antigenic source for the excess production of anti-Gal. In view of the naturally high level of anti-Gal in humans and its binding to T. cruzi and Leishmania, it is argued that these antibodies may contribute to the natural defense against the invasion of such parasites.

  16. Biosynthesis of alpha2(IV) and alpha1(IV) chains of collagen IV and interactions with matrix metalloproteinase-9.

    PubMed

    Toth, M; Sado, Y; Ninomiya, Y; Fridman, R

    1999-07-01

    In vitro binding studies with latent matrix metalloproteinase-9 (pro-MMP-9) have revealed the existence of nondisulfide-bonded alpha2(IV) chains on the cell surface capable of forming a high-affinity complex with the enzyme. Here we investigated the biosynthesis and cellular distribution of alpha2(IV) and alpha1(IV) chains in breast epithelial (MCF10A and MDA-MB-231) and fibrosarcoma (HT1080) cells by pulse-chase analysis followed by immunoprecipitation with chain-specific monoclonal antibodies (mAb). These studies showed that whereas the alpha1(IV) chain remained in the intracellular compartment, nondisulfide-bonded alpha2(IV) chains were secreted into the media in a stable form. Consistently, only alpha2(IV) was detected on the cell surface by surface biotinylation or indirect immunofluorescence. In agreement with the pulse-chase analysis, media subjected to co-precipitation experiments with pro-MMP-9 or pro-MMP-9-affinity chromatography followed by immunoblotting with chain-specific mAbs resulted in the detection of alpha2(IV). A preferential secretion of nondisulfide-bonded alpha2(IV) chains was also observed in CHO-K1 cells transiently transfected with full-length mouse alpha2(IV) or alpha (IV) cDNAs. However, a complex of mouse alpha1(IV) with pro-MMP-9 was coprecipitated with exogenous enzyme from lysates of CHO-K1 cells transfected with mouse alpha1(IV), suggesting that under overexpression conditions the enzyme can also interact with the alpha1 (IV) chain. Collectively, these studies further demonstrate the interactions of pro-MMP-9 with collagen IV chains and a unique processing and targeting of nondisulfide-bonded alpha2(IV) chains that may play a role in the surface/matrix association of pro-MMP-9.

  17. Alpha-1-adrenergic modulation of K and Cl transport in bovine retinal pigment epithelium

    PubMed Central

    1992-01-01

    Intracellular microelectrode techniques were used to characterize the electrical responses of the bovine retinal pigment epithelium (RPE)- choroid to epinephrine (EP) and several other catecholamines that are putative paracrine signals between the neural retina and the RPE. Nanomolar amounts of EP or norepinephrine (NEP), added to the apical bath, caused a series of conductance and voltage changes, first at the basolateral or choroid-facing membrane and then at the apical or retina- facing membrane. The relative potency of several adrenergic agonists and antagonists indicates that EP modulation of RPE transport begins with the activation of apical alpha-1-adrenergic receptors. The membrane-permeable calcium (Ca2+) buffer, amyl-BAPTA (1,2-bis(o- aminophenoxy)-ethane-N,N,N',N' tetraacetic acid) inhibited the EP- induced voltage and conductance changes by approximately 50-80%, implicating [Ca2+]i as a second messenger. This conclusion is supported by experiments using the Ca2+ ionophore A23187, which mimics the effects of EP. The basolateral membrane voltage response to EP was blocked by lowering cell Cl, by the presence of DIDS (4,4'- diisothiocyanostilbene-2,2'-disulfonic acid) in the basal bath, and by current clamping VB to the Cl equilibrium potential. In the latter experiments the EP-induced conductance changes were unaltered, indicating that EP increases basolateral membrane Cl conductance independent of voltage. The EP-induced change in basolateral Cl conductance was followed by a secondary decrease in apical membrane K conductance (approximately 50%) as measured by delta [K]o-induced diffusion potentials. Decreasing apical K from 5 to 2 mM in the presence of EP mimicked the effect of light on RPE apical and basolateral membrane voltage. These results indicate that EP may be an important paracrine signal that provides exquisite control of RPE physiology. PMID:1319462

  18. Alpha(1)-adrenergic-mediated eNOS phosphorylation in intact arteries.

    PubMed

    Looft-Wilson, Robin C; Todd, Sarah E; Araj, Christina A; Mutchler, Stephanie M; Goodell, Cara A Raphael

    2013-01-01

    Activation of arterial smooth muscle alpha(1)-adrenergic receptors results in vasoconstriction, as well as a secondary release of nitric oxide and slow vasodilation, presumably through gap junction communication from smooth muscle to endothelium. We hypothesized that this slow vasodilation is due to activation of eNOS through phosphorylation at Ser1179 and dephosphorylation at Thr495. Phosphorylation was measured by western blot using mouse mesenteric arteries that were cannulated and pressurized (75 mm Hg) and treated either by 1) 5 min of phenylephrine superfusion (10(-5)M) (PE5), 2) 15 min of phenylephrine (PE15), 3) 15 min phenylephrine followed by acetylcholine (10(-4)M) (PE+ACh), or 4) 20 min time control with no treatment (NT) [4-5 arteries pooled per treatment per blot; 5 blots performed]. These treatments allowed correlation between vasomotor changes, namely maximal constriction (PE5), slow vasodilation (PE15), and maximal dilation (PE+ACh), and relative phosphorylation changes. Phosphorylation of eNOS at Ser1179 was increased relative to NT by more than 2-fold at PE5 and remained similarly increased at PE15 and PE+ACh. Phosphorylation of eNOS at Thr495 was less in all treatments relative to NT, but not significantly. Treatment with L-NAME (10(-4)M) or endothelial denudation indicated that the slow dilation in response to phenylephrine was completely due to nitric oxide synthase and was endothelial dependent. These results indicate that eNOS phosphorylation at Ser1179 occurs before the slow dilation and is not actively involved in this vasodilation or dilation to acetylcholine, but may play a permissive role in eNOS activation by other mechanisms. It is not yet known what mechanism is responsible for Ser1179 phosphorylation with phenylephrine stimulation.

  19. Alpha-1 adrenoceptor blockade with doxazosin in hypertension: effects on blood pressure and lipoproteins.

    PubMed

    Ames, R P; Kiyasu, J Y

    1989-02-01

    The effects of doxazosin, a long-acting alpha-1 adrenoreceptor blocking drug, were observed upon blood pressure and serum lipoproteins. Thirty patients with supine diastolic blood pressure between 90 and 114 mm Hg during single-blind placebo therapy were randomized to double-blind treatment with either doxazosin or further placebo in a parallel-design protocol. Starting at one mg, dosage was doubled every 2 weeks during a 10-week treatment period to a maximum dose of 16 mg once daily. Blood was sampled in the fasting state before and during double-blind therapy for measurement of total cholesterol and triglycerides, cholesterol in the lipoprotein fractions, and apolipoproteins A and B. At the end of 10 weeks of titration, systolic and diastolic blood pressure were each reduced by 14 mm Hg in the standing position when measured 24 hours following the previous dose. Supine pressure was lowered by 6 mm Hg systolic and by 5 mm Hg diastolic at the same time point. Measured hourly for 12 hours following the ingestion of doxazosin, blood pressure was lowered maximally at 4-5 hours when an additional decline of 14/6 mm Hg (systolic/diastolic) was observed in the standing position and 13/6 in the supine posture. Postural dizziness, the most frequent symptomatic complaint, was reported in 4 patients during doxazosin treatment. After brief interruption of treatment in one and dosage adjustment in another, titration was continued in all four and no patient was withdrawn because of side effects. Concerning lipoproteins, the ratio of total cholesterol to HDL cholesterol and of LDL to HDL cholesterol both improved during treatment with doxazosin.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. (-)-Discretamine, a selective alpha 1D-adrenoceptor antagonist, isolated from Fissistigma glaucescens.

    PubMed Central

    Ko, F. N.; Guh, J. H.; Yu, S. M.; Hou, Y. S.; Wu, Y. C.; Teng, C. M.

    1994-01-01

    1. The selectivity of (-)-discretamine for alpha 1-adrenoceptor subtypes was investigated by use of functional and binding studies in rat vas deferens, spleen and aorta, and in cultured DDT1MF-2 and A10 cells. 2. In prostatic portions of rat vas deferens, the competitive antagonists (-)-discretamine, 5-methylurapidil (5-MU) and prazosin inhibited contractions to noradrenaline (NA) with pA2 values of 6.21, 8.71 and 9.27, respectively. The irreversible antagonist, chloroethylclonidine (CEC, 100 microM) failed to affect contractions to NA while nifedipine (1 microM) blocked them almost completely. 3. In rat spleen, the competitive antagonists (-)-discretamine, 5-MU and prazosin inhibited contractions to phenylephrine with pA2 values of 6.44, 7.19 and 9.45, respectively. CEC (100 microM) significantly reduced the maximum contraction to phenylephrine while nifedipine (1 microM) did not affect it. 4. In rat aorta, the competitive antagonists (-)-discretamine, 5-MU and prazosin inhibited contractions to NA with pA2 values of 7.60, 8.00 and 9.40, respectively. CEC also antagonized the contractions to NA in a competitive manner with a pA2 value of 6.10. 5. The specific binding of [3H]-prazosin to DDT1MF-2 and A10 cells was concentration-dependent and saturated at 3-5 nM with KD values of 0.24 +/- 0.02 and 0.20 +/- 0.02 nM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7952879

  1. Distribution of primaquine in human blood: Drug-binding to alpha 1-glycoprotein

    SciTech Connect

    Kennedy, E.; Frischer, H. )

    1990-12-01

    To clarify the distribution of the antimalarial primaquine in human blood, we measured the drug separately in the liquid, cellular, and ultrafiltrate phases. Washed red cells resuspended at a hematocrit of 0.4 were exposed to a submaximal therapeutic level of 250 ng/ml of carbon 14-labeled primaquine. The tracer was recovered quantitatively in separated plasma and red cells. Over 75% of the total labeled drug was found in red cells suspended in saline solution, but only 10% to 30% in red cells suspended in plasma. The plasma effect was not mediated by albumin. Studies with alpha 1-acid glycoprotein (AGP), tris(2-butoxyethyl)phosphate, an agent that displaces AGP-bound drugs, and cord blood known to have decreased AGP established that primaquine binds to physiologic amounts of the glycoprotein in plasma. Red cell primaquine concentration increased linearly as AGP level fell and as the free drug fraction rose. We suggest that clinical blood levels of primaquine include the red cell fraction or whole blood level because (1) erythrocytic primaquine is a sizable and highly variable component of the total drug in blood; (2) this component reflects directly the free drug in plasma, and inversely the extent of binding to AGP; (3) the amount of free primaquine may influence drug transport into specific tissues in vivo; and (4) fluctuations of AGP, an acute-phase reactant that increases greatly in patients with malaria and other infections, markedly affect the partition of primaquine in blood. Because AGP binds many basic drugs, unrecognized primaquine-drug interactions may exist.

  2. Transcriptional promoter of the human alpha 1(V) collagen gene (COL5A1).

    PubMed Central

    Lee, S; Greenspan, D S

    1995-01-01

    We have characterized the 5' region of the human alpha 1(V) collagen gene (COL5A1). The transcriptional promoter is shown to have a number of features characteristic of the promoters of 'housekeeping' and growth-control-related genes. It lacks obvious TATA and CAAT boxes, has multiple transcription start sites, has a high GC content, lies within a well-defined CpG island and has a number of consensus sites for the potential binding of transcription factor Sp1. This type of promoter structure, while unusual for a collagen gene, is consistent with the broad distribution of expression of COL5A1 and is reminiscent of the promoter structures of the genes encoding type VI collagen, which has a similarly broad distribution of expression. Stepwise deletion of COL5A1 5' sequences, placed upstream of a heterologous reporter gene, yielded a gradual decrease in promoter activity, indicating that the COL5A1 promoter is composed of an array of cis-acting elements. A minimal promoter region contained within the 212 bp immediately upstream of the major transcription start site contained no consensus sequences for the binding of known transcription factors, but gel mobility shift assays showed this region to bind nuclear factors, including Sp1, at a number of sites. The major transcription start site is flanked by an upstream 34-bp oligopurine/oligopyrimidine stretch, or 'GAGA' box, and a downstream 56-bp GAGA box which contains a 10-bp mirror repeat and is sensitive to cleavage with S1 nuclease. Images Figure 1 Figure 3 Figure 4 Figure 6 PMID:7646438

  3. Multilineage transduction of resident lung cells in vivo by AAV2/8 for α1-antitrypsin gene therapy

    PubMed Central

    Payne, Julia G; Takahashi, Ayuko; Higgins, Michelle I; Porter, Emily L; Suki, Bela; Balazs, Alejandro; Wilson, Andrew A

    2016-01-01

    In vivo gene delivery has long represented an appealing potential treatment approach for monogenic diseases such as α1-antitrypsin deficiency (AATD) but has proven challenging to achieve in practice. Alternate pseudotyping of recombinant adeno-associated virus (AAV) vectors is producing vectors with increasingly heterogeneous tropic specificity, giving researchers the ability to target numerous end-organs affected by disease. Herein, we describe sustained pulmonary transgene expression for at least 52 weeks after a single intratracheal instillation of AAV2/8 and characterize the multiple cell types transduced within the lung utilizing this approach. We demonstrate that lung-directed AAV2/8 is able to achieve therapeutic α-1 antitrypsin (AAT) protein levels within the lung epithelial lining fluid and that AAT gene delivery ameliorates the severity of experimental emphysema in mice. We find that AAV2/8 efficiently transduces hepatocytes in vivo after intratracheal administration, a finding that may have significance for AAV-based human gene therapy studies. These results support direct transgene delivery to the lung as a potential alternative approach to achieve the goal of developing a gene therapy for AATD. PMID:27408904

  4. Multilineage transduction of resident lung cells in vivo by AAV2/8 for α1-antitrypsin gene therapy.

    PubMed

    Payne, Julia G; Takahashi, Ayuko; Higgins, Michelle I; Porter, Emily L; Suki, Bela; Balazs, Alejandro; Wilson, Andrew A

    2016-01-01

    In vivo gene delivery has long represented an appealing potential treatment approach for monogenic diseases such as α1-antitrypsin deficiency (AATD) but has proven challenging to achieve in practice. Alternate pseudotyping of recombinant adeno-associated virus (AAV) vectors is producing vectors with increasingly heterogeneous tropic specificity, giving researchers the ability to target numerous end-organs affected by disease. Herein, we describe sustained pulmonary transgene expression for at least 52 weeks after a single intratracheal instillation of AAV2/8 and characterize the multiple cell types transduced within the lung utilizing this approach. We demonstrate that lung-directed AAV2/8 is able to achieve therapeutic α-1 antitrypsin (AAT) protein levels within the lung epithelial lining fluid and that AAT gene delivery ameliorates the severity of experimental emphysema in mice. We find that AAV2/8 efficiently transduces hepatocytes in vivo after intratracheal administration, a finding that may have significance for AAV-based human gene therapy studies. These results support direct transgene delivery to the lung as a potential alternative approach to achieve the goal of developing a gene therapy for AATD. PMID:27408904

  5. A genome-wide RNAi screen identifies potential drug targets in a C. elegans model of α1-antitrypsin deficiency

    PubMed Central

    O'Reilly, Linda P.; Long, Olivia S.; Cobanoglu, Murat C.; Benson, Joshua A.; Luke, Cliff J.; Miedel, Mark T.; Hale, Pamela; Perlmutter, David H.; Bahar, Ivet; Silverman, Gary A.; Pak, Stephen C.

    2014-01-01

    α1-Antitrypsin deficiency (ATD) is a common genetic disorder that can lead to end-stage liver and lung disease. Although liver transplantation remains the only therapy currently available, manipulation of the proteostasis network (PN) by small molecule therapeutics offers great promise. To accelerate the drug-discovery process for this disease, we first developed a semi-automated high-throughput/content-genome-wide RNAi screen to identify PN modifiers affecting the accumulation of the α1-antitrypsin Z mutant (ATZ) in a Caenorhabditis elegans model of ATD. We identified 104 PN modifiers, and these genes were used in a computational strategy to identify human ortholog–ligand pairs. Based on rigorous selection criteria, we identified four FDA-approved drugs directed against four different PN targets that decreased the accumulation of ATZ in C. elegans. We also tested one of the compounds in a mammalian cell line with similar results. This methodology also proved useful in confirming drug targets in vivo, and predicting the success of combination therapy. We propose that small animal models of genetic disorders combined with genome-wide RNAi screening and computational methods can be used to rapidly, economically and strategically prime the preclinical discovery pipeline for rare and neglected diseases with limited therapeutic options. PMID:24838285

  6. α-1-Antitrypsin detected by MALDI imaging in the study of glomerulonephritis: Its relevance in chronic kidney disease progression.

    PubMed

    Smith, Andrew; L'Imperio, Vincenzo; De Sio, Gabriele; Ferrario, Franco; Scalia, Carla; Dell'Antonio, Giacomo; Pieruzzi, Federico; Pontillo, Claudia; Filip, Szymon; Markoska, Katerina; Granata, Antonio; Spasovski, Goce; Jankowski, Joachim; Capasso, Giovambattista; Pagni, Fabio; Magni, Fulvio

    2016-06-01

    Idiopathic glomerulonephritis (GN), such as membranous glomerulonephritis, focal segmental glomerulosclerosis (FSGS), and IgA nephropathy (IgAN), represent the most frequent primary glomerular kidney diseases (GKDs) worldwide. Although the renal biopsy currently remains the gold standard for the routine diagnosis of idiopathic GN, the invasiveness and diagnostic difficulty related with this procedure highlight the strong need for new diagnostic and prognostic biomarkers to be translated into less invasive diagnostic tools. MALDI-MS imaging MALDI-MSI was applied to fresh-frozen bioptic renal tissue from patients with a histological diagnosis of FSGS (n = 6), IgAN, (n = 6) and membranous glomerulonephritis (n = 7), and from controls (n = 4) in order to detect specific molecular signatures of primary glomerulonephritis. MALDI-MSI was able to generate molecular signatures capable to distinguish between normal kidney and pathological GN, with specific signals (m/z 4025, 4048, and 4963) representing potential indicators of chronic kidney disease development. Moreover, specific disease-related signatures (m/z 4025 and 4048 for FSGS, m/z 4963 and 5072 for IgAN) were detected. Of these signals, m/z 4048 was identified as α-1-antitrypsin and was shown to be localized to the podocytes within sclerotic glomeruli by immunohistochemistry. α-1-Antitrypsin could be one of the markers of podocyte stress that is correlated with the development of FSGS due to both an excessive loss and a hypertrophy of podocytes.

  7. Structures of NodZ [alpha]1,6-fucosyltransferase in complex with GDP and GDP-fucose

    SciTech Connect

    Brzezinski, Krzysztof; Dauter, Zbigniew; Jaskolski, Mariusz

    2012-03-26

    Rhizobial NodZ {alpha}1,6-fucosyltransferase ({alpha}1,6-FucT) catalyzes the transfer of the fucose (Fuc) moiety from guanosine 5'-diphosphate-{beta}-L-fucose to the reducing end of the chitin oligosaccharide core during Nod-factor (NF) biosynthesis. NF is a key signaling molecule required for successful symbiosis with a legume host for atmospheric nitrogen fixation. To date, only two {alpha}1,6-FucT structures have been determined, both without any donor or acceptor molecule that could highlight the structural background of the catalytic mechanism. Here, the first crystal structures of {alpha}1,6-FucT in complex with its substrate GDP-Fuc and with GDP, which is a byproduct of the enzymatic reaction, are presented. The crystal of the complex with GDP-Fuc was obtained through soaking of native NodZ crystals with the ligand and its structure has been determined at 2.35 {angstrom} resolution. The fucose residue is exposed to solvent and is disordered. The enzyme-product complex crystal was obtained by cocrystallization with GDP and an acceptor molecule, penta-N-acetyl-L-glucosamine (penta-NAG). The structure has been determined at 1.98 {angstrom} resolution, showing that only the GDP molecule is present in the complex. In both structures the ligands are located in a cleft formed between the two domains of NodZ and extend towards the C-terminal domain, but their conformations differ significantly. The structures revealed that residues in three regions of the C-terminal domain, which are conserved among {alpha}1,2-, {alpha}1,6- and protein O-fucosyltransferases, are involved in interactions with the sugar-donor molecule. There is also an interaction with the side chain of Tyr45 in the N-terminal domain, which is very unusual for a GT-B-type glycosyltransferase. Only minor conformational changes of the protein backbone are observed upon ligand binding. The only exception is a movement of the loop located between strand {beta}C2 and helix {alpha}C3. In addition, there is

  8. Physical and genetic mapping of the serpin gene cluster at 14q32. 1: Allelic association and a unique haplotype associated with [alpha][sub 1]-antitrypsin deficiency

    SciTech Connect

    Byth, B.C.; Billingsley, G.D.; Cox, D.W. )

    1994-07-01

    The [alpha][sub 1]-antitrypsin (PI) gene is part of a cluster of structurally related serine protease inhibitor genes localized at chromosome 14q32, a cluster that includes the [alpha][sub 1]-anticymotrypsin (AACT), protein C inhibitor (PCI), and corticosteroid-binding globulin (CBG) genes and the [alpha][sub 1]-antitrypsin-like pseudogene (PIL). The order of the genes is refined here by genetic mapping using simple tandem repeat polymorphisms (STRPs) and by physical mapping in YACs. The order of the genes is (cetromere)-CBG-PIL-PI-PCI-AACT-(telomere). Analysis of DNA haplotypes comprising STRP and RFLP markers in the serpin genes reveals considerable allelic association throughout the cluster. Furthermore, the common [alpha][sub 1]-antitrypsin deficiency allele, PI[sup *]Z, has a unique DNA haplotype at the CBG, PIL, and PI loci, which extends over 60 kb in 97% of cases and in 44% of cases includes the PCI and AACT loci. This unique haplotype will be of use in examining a number of other diseases, particularly those with an inflammatory component, thought to be associated with [alpha][sub 1]-antitrypsin deficiency or partial deficiency. 23 refs., 6 figs., 4 tabs.

  9. Contractions mediated by alpha 1-adrenoceptors and P2-purinoceptors in a cat colon circular muscle.

    PubMed Central

    Venkova, K.; Milne, A.; Krier, J.

    1994-01-01

    1. The postjunctional excitatory and inhibitory effects of adrenoceptor and purinoceptor agonists and antagonists were studied in circular smooth muscle strips of cat colon. 2. In the presence of tetrodotoxin (0.5 microM), noradrenaline caused contraction or relaxation of circular smooth muscle at resting tension or with raised tone, respectively. The noradrenaline-evoked contractions were potentiated and the noradrenaline-evoked relaxations were antagonized by propranolol (1 microM), suggesting beta-adrenoceptor involvement. 3. At resting tension, noradrenaline, adrenaline and the selective alpha 1-adrenoceptor agonist, phenylephrine, caused concentration-dependent contractile responses, with EC50 values of 1.8 +/- 0.2 microM, 1.9 +/- 0.4 microM and 4.3 +/- 1.7 microM, respectively. The EC50 values and the amplitude of maximal responses were not significantly different from one another. Clonidine (0.1-500 microM), a selective alpha 2-adrenoceptor agonist, was not effective. 4. Prazosin (0.1-9 microM), competitively antagonized the contractile effects of noradrenaline with an estimated pA2 value of 6.93 and a slope of 1.07 +/- 0.03. The Kb values, estimated from a single shift (0.1 microM prazosin) of the concentration-response curves to noradrenaline, adrenaline and phenylephrine were 92.8 +/- 9.3 nM, 108.7 +/- 6.4 nM and 18.4 +/- 3.1 nM, respectively. 5. At resting tension, adenosine 5' triphosphate (ATP, 5-1000 microM), alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-MeATP, 0.05-50 microM), beta,gamma-methylene adenosine 5'-triphosphate (beta,gamma-MeATP, 0.5-100 microM), and 2-methylthioadenosine 5'-triphosphate (2-MeSATP, 1-500 microM) caused concentration-dependent contractions with EC50 values of 60.5 +/- 15.9 microM, 0.7 +/- 0.1 microM, 7.6 +/- 0.1 microM and 25.3 +/- 12.8 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7952886

  10. Alpha1-antichymotrypsin, an inflammatory protein overexpressed in Alzheimer's disease brain, induces tau phosphorylation in neurons.

    PubMed

    Padmanabhan, Jaya; Levy, Monique; Dickson, Dennis W; Potter, Huntington

    2006-11-01

    Amyloid plaques and neurofibrillary tangles are key pathological features of Alzheimer's disease. Alzheimer's disease pathology is also characterized by neuroinflammation and neuronal degeneration, with the proteins associated with inflammatory responses being found in tight association with the plaques. One such protein is the serine protease inhibitor alpha-1-antichymotrypsin (ACT). ACT has been shown to promote Abeta polymerization in vitro and in vivo, and levels of ACT protein in plasma and cerebrospinal fluid from Alzheimer's patients have been found to correlate with progression of dementia. Here we investigated the possible involvement of ACT in tau phosphorylation and tangle formation. As was previously found for Alzheimer's disease, brains from patients with non-Alzheimer's tauopathies exhibited an enhanced expression of ACT, which correlated with the level of tau hyperphosphorylation. Transgenic mice expressing human ACT alone or ACT along with mutant human amyloid precursor protein (APP) showed a significant increase in tau phosphorylation, suggesting that this inflammatory protein can induce tau hyperphosphorylation. The increase in phosphorylation was observed at PHF-1 (P-Ser396/P-Thr404), P-Ser202 and P-Thr231 sites on tau, the P-tau epitopes that are associated with tangles in the patients. This result was further confirmed by the finding that addition of purified ACT induced the same Alzheimer's disease-related tau hyperphosphorylation in cortical neurons cultured in vitro. This correlated with an increase in extracellular signal regulated kinase (ERK) and glycogen synthase kinase-3 activation, indicating their involvement in ACT-induced tau phosphorylation. The ACT-treated neurons showed neurite loss and subsequently underwent apoptosis. Approximately 40-50% of neurons were TUNEL positive by 6 and at 24 h >70% of the neurons showed staining suggesting that ACT was inducing apoptosis in these neurons. These findings indicate that inappropriate

  11. Serum concentrations of canine alpha(1)-proteinase inhibitor in cobalamin-deficient Yorkshire Terrier dogs.

    PubMed

    Grützner, Niels; Heilmann, Romy M; Bridges, Cory S; Suchodolski, Jan S; Steiner, Jörg M

    2013-05-01

    Fecal canine alpha1-proteinase inhibitor (cα1-PI) concentration has been reported to be increased in dogs with protein-losing enteropathy due to the loss of cα1-PI into the gastrointestinal tract. A chronic loss of cα1-PI may theoretically deplete serum cα1-PI, potentially altering the proteinase-to-proteinase inhibitor balance. Protein-losing enteropathy has been reported to occur frequently in certain dog breeds such as Yorkshire Terriers and to be associated with hypocobalaminemia. The objective was to compare serum cα1-PI concentrations in Yorkshire Terriers with and without cobalamin (COB) deficiency. Serum samples from 52 COB-deficient and 69 normocobalaminemic Yorkshire Terriers, which had been submitted to the Gastrointestinal Laboratory (2008-2011; College Station, TX), were included retrospectively. Serum cα1-PI concentrations were measured using an in-house radioimmunoassay and compared between Yorkshire Terriers with and without COB deficiency using a Mann-Whitney U test. A Fisher exact test was used to evaluate whether a decreased serum cα1-PI concentration is associated with COB deficiency in Yorkshire Terriers. Serum cα1-PI concentrations were significantly lower in COB-deficient Yorkshire Terriers (median: 1,016 mg/l, range: 315-3,945 mg/l) than in normocobalaminemic Yorkshire Terriers (median: 1,665 mg/l, range: 900-2,970 mg/l; P < 0.0001). One-fourth (n = 13) of the COB-deficient Yorkshire Terriers had a serum cα1-PI concentration below the lower limit of the reference interval (<732 mg/l), and COB deficiency was associated with decreased serum cα1-PI concentrations (P < 0.0001). In the current study, serum cα1-PI concentrations are significantly lower in COB-deficient Yorkshire Terriers when compared to normocobalaminemic Yorkshire Terriers. Further studies are needed to determine the functional and potential prognostic implications of serum cα1-PI concentrations in dogs with gastrointestinal disease.

  12. alpha1-Adrenoceptors stimulate a Galphas protein and reduce the transient outward K+ current via a cAMP/PKA-mediated pathway in the rat heart.

    PubMed

    Gallego, Mónica; Setién, Raúl; Puebla, Lilian; Boyano-Adánez, María Del Carmen; Arilla, Eduardo; Casis, Oscar

    2005-03-01

    alpha(1)-Adrenoceptor stimulation prolongs the duration of the cardiac action potentials and leads to positive inotropic effects by inhibiting the transient outward K(+) current (I(to)). In the present study, we have examined the role of several protein kinases and the G protein involved in I(to) inhibition in response to alpha(1)-adrenoceptor stimulation in isolated adult rat ventricular myocytes. Our findings exclude the classic alpha(1)-adrenergic pathway: activation of the G protein G(alphaq), phospholipase C (PLC), and protein kinase C (PKC), because neither PLC, nor PKC, nor G(alphaq) blockade prevents the alpha(1)-induced I(to) reduction. To the contrary, the alpha(1)-adrenoceptor does not inhibit I(to) in the presence of protein kinase A (PKA), adenylyl cyclase, or G(alphas) inhibitors. In addition, PKA and adenylyl cyclase activation inhibit I(to) to the same extent as phenylephrine. Finally, we have shown a functional coupling between the alpha(1)-adrenoceptor and G(alphas) in a physiological system. Moreover, this coupling seems to be compartmentalized, because the alpha(1)-adrenoceptor increases cAMP levels only in intact cells, but not in isolated membranes, and the effect on I(to) disappears when the cytoskeleton is disrupted. We conclude that alpha(1)-adrenoceptor stimulation reduces the amplitude of the I(to) by activating a G(alphas) protein and the cAMP/PKA signaling cascade, which in turn leads to I(to) channel phosphorylation.

  13. A small segment of the MAT alpha 1 transcript promotes mRNA decay in Saccharomyces cerevisiae: a stimulatory role for rare codons.

    PubMed Central

    Caponigro, G; Muhlrad, D; Parker, R

    1993-01-01

    Differences in decay rates of eukaryotic transcripts can be determined by discrete sequence elements within mRNAs. Through the analysis of chimeric transcripts and internal deletions, we have identified a 65-nucleotide segment of the MAT alpha 1 mRNA coding region, termed the MAT alpha 1 instability element, that is sufficient to confer instability to a stable PGK1 reporter transcript and that accelerates turnover of the unstable MAT alpha 1 mRNA. This 65-nucleotide element is composed of two parts, one located within the 5' 33 nucleotides and the second located in the 3' 32 nucleotides. The first part, which can be functionally replaced by sequences containing rare codons, is unable to promote rapid decay by itself but can enhance the action of the 3' 32 nucleotides (positions 234 to 266 in the MAT alpha 1 mRNA) in accelerating turnover. A second portion of the MAT alpha 1 mRNA (nucleotides 265 to 290) is also sufficient to destabilize the PGK1 reporter transcript when positioned 3' of rare codons, suggesting that the 3' half of the MAT alpha 1 instability element is functionally reiterated within the MAT alpha 1 mRNA. The observation that rare codons are part of the 65-nucleotide MAT alpha 1 instability element suggests possible mechanisms through which translation and mRNA decay may be linked. Images PMID:8355674

  14. Sensitization of rhabdo-, lenti-, and spumaviruses to human serum by galactosyl(alpha1-3)galactosylation.

    PubMed

    Takeuchi, Y; Liong, S H; Bieniasz, P D; Jäger, U; Porter, C D; Friedman, T; McClure, M O; Weiss, R A

    1997-08-01

    Vesicular stomatitis virus, human immunodeficiency virus type 2, and human foamy virus, which were produced by cell lines expressing galactosyl(alpha1-3)galactosyl (alphaGal) sugars, were found to be less stable in human serum than those from alphaGal-negative cells, indicating that galactosyl(alpha1-3)galactosylation sensitizes these viruses as well as mammalian type C oncoviruses (Rother et al., J. Exp. Med. 182:1345-1355, 1995; Takeuchi et al., Nature (London) 379:85-88, 1996) to complement killing via natural anti-alphaGal antibodies. Thus, virus killing mediated by anti-alphaGal antibodies may play a role as a barrier to animal-to-human infection of various enveloped viruses. Virus vectors for human in vivo gene therapy based on the viruses mentioned above should be produced from alphaGal-negative cells. PMID:9223512

  15. High-density lipoproteins potentiate α1-antitrypsin therapy in elastase-induced pulmonary emphysema.

    PubMed

    Moreno, Juan-Antonio; Ortega-Gomez, Almudena; Rubio-Navarro, Alfonso; Louedec, Liliane; Ho-Tin-Noé, Benoit; Caligiuri, Giuseppina; Nicoletti, Antonino; Levoye, Angelique; Plantier, Laurent; Meilhac, Olivier

    2014-10-01

    Several studies report that high-density lipoproteins (HDLs) can carry α1-antitrypsin (AAT; an elastase inhibitor). We aimed to determine whether injection of exogenous HDL, enriched or not in AAT, may have protective effects against pulmonary emphysema. After tracheal instillation of saline or elastase, mice were randomly treated intravenously with saline, human plasma HDL (75 mg apolipoprotein A1/kg), HDL-AAT (75 mg apolipoprotein A1-3.75 mg AAT/kg), or AAT alone (3.75 mg/kg) at 2, 24, 48, and 72 hours. We have shown that HDL-AAT reached the lung and prevented the development of pulmonary emphysema by 59.3% at 3 weeks (alveoli mean chord length, 22.9 ± 2.8 μm versus 30.7 ± 4.5 μm; P < 0.001), whereas injection of HDL or AAT alone only showed a moderate, nonsignificant protective effect (28.2 ± 4.2 μm versus 30.7 ± 5 μm [P = 0.23] and 27.3 ± 5.66 μm versus 30.71 ± 4.96 μm [P = 0.18], respectively). Indeed, protection by HDL-AAT was significantly higher than that observed with HDL or AAT (P = 0.006 and P = 0.048, respectively). This protective effect was associated (at 6, 24, and 72 h) with: (1) a reduction in neutrophil and macrophage number in the bronchoalveolar lavage fluid; (2) decreased concentrations of IL-6, monocyte chemoattractant protein-1, and TNF-α in both bronchoalveolar lavage fluid and plasma; (3) a reduction in matrix metalloproteinase-2 and matrix metalloproteinase-9 activities; and (4) a reduction in the degradation of fibronectin, a marker of tissue damage. In addition, HDL-AAT reduced acute cigarette smoke-induced inflammatory response. Intravenous HDL-AAT treatment afforded a better protection against elastase-induced pulmonary emphysema than AAT alone, and may represent a significant development for the management of emphysema associated with AAT deficiency.

  16. Tissue-specific loss of fucosylated glycolipids in mice with targeted deletion of alpha(1,2)fucosyltransferase genes.

    PubMed Central

    Iwamori, Masao; Domino, Steven E

    2004-01-01

    Glycolipids in epithelial tissues of the gastrointestinal tract act as receptors for enteric bacteria and are implicated in the activation of the intestinal immune system. To clarify the genes involved in the fucosylation of the major glycolipids, substrate glycolipids and fucosylated products were measured in tissues of wild-type and mutant mice lacking alpha(1,2)fucosyltransferase genes FUT1 or FUT2. Quantitative determination was performed by TLC-immunostaining for GA1 (Gg4Cer), FGA1 (fucosyl GA1), GM1 (II3NeuAc-Gg4Cer), FGM1 (fucosyl GM1), and Forssman glycolipids. Both FGM1 and FGA1 completely disappeared from the antrum, cecum, and colon of FUT2-null mice, but not those of FUT1-null and wild-type mice. Precursor glycolipids, GM1 and GA1, accumulated in tissues of FUT2-null mice, indicating that the FUT2-encoded enzyme preferentially participates in the fucosylation of GA1 and GM1 in these tissues. Female reproductive organs were similarly found to utilize FUT2 for the fucosylation of glycolipids FGA1 (uterus and cervix), and FGM1 (ovary), due to their absence in FUT2-null mice. In FUT1-null mice FGA1 was lost from the pancreas, but was present in wild-type and FUT2-null mice, indicating that FUT1 is essential for fucosylation of GA1 in the pancreas. Ulex europaeus agglutinin-I lectin histochemistry for alpha(1,2)fucose residues confirmed the absence of alpha(1,2)fucose residues from the apical surface of pancreatic acinar glands of FUT1-null mice. Ileum, epididymis, and testis retained specific fucosylated glycolipids, irrespective of targeted deletion of either gene, indicating either compensation for or redundancy of the alpha(1,2)fucosyltransferase genes in these tissues. PMID:14967068

  17. The use of alpha-1 adrenergic blockers in children with distal ureterolithiasis: a systematic review and meta-analysis

    PubMed Central

    Glina, F.P.; Castro, P.M.V.; Monteiro, G.G.R.; Guerra, G.C. Del; Glina, S.; Mazzurana, M.; Bernardo, W.M.

    2015-01-01

    ABSTRACT Introduction: Urinary lithiasis is the main urologic cause of emergency treatment in adult patient. In the past years, the incidence in children population has increased. However, literature about the use of alpha-1 adrenergic blockers in pediatric population with distal ureterolithiasis is still scarce. The drug acts by decreasing ureter contractions, especially in the distal portion, facilitating calculus expulsion. Objective: This review has the objective to evaluate the use of alpha-1 adrenergic blockers as medical expulsive treatment in children with distal ureterolithiasis. Evidence Acquisition: An electronic literature search was performed using the MEDLINE, COCHRANE, and LILACS databases. We further searched manually the references of the primary studies. Searches were concluded on October 4th, 2014. Articles were selected, independently and in pairs, by the respective titles and summaries. Any divergence was resolved by consensus. Evidence Synthesis: Alpha-1 adrenergic antagonists increased the probability of calculus expulsion by 27% (NNT=4). Calculi smaller than 5mm, increased by 33% (NNT=3). Larger than 5mm, increased by 34% (NNT=3). Conclusion: Alpha-1 adrenergic blocker use is related with a greater incidence of expulsion of ureteral calculi, smaller or greater than 5mm, and fewer episodes of pain when compared to ibuprofen. However it is necessary larger samples to enhance the power analysis of the expulsion of ureteral calculi larger than 5mm and the episodes of pain. Patient Summary: This review analyzed the outcome of alpha adrenergic antagonist in children with ureteral calculi. We conclude that it is the best medicine for use, since it helps the expulsion of the stone. PMID:26717117

  18. Effects of jump training on procollagen alpha(1)(i) mRNA expression and its relationship with muscle collagen concentration.

    PubMed

    Ducomps, Christophe; Larrouy, Dominique; Mairal, Aline; Doutreloux, Jean-Paul; Lebas, Francois; Mauriege, Pascale

    2004-04-01

    The aim of this study was to examine the effects of a prolonged high-intensity exercise, jumping, on procollagen alpha(1)(I) mRNA level and collagen concentration in different muscles of trained (T) and control (C) rabbits. Procollagen alpha(1)(I) mRNA expression was much higher (2.8 to 23.5 times) in semimembranosus proprius (SMP), a slow-twitch oxidative muscle, than in extensor digitorum longus (EDL), rectus femoris (RF), and psoas major (Psoas) muscles, both fast-twitch mixed and glycolytic, whatever group was considered (p < 0.001). Procollagen alpha(1)(I) mRNA level also decreased significantly between 50 and 140 days in all muscles (0.001< p < 0.01). However, mRNA levels were 16 to 97% greater at 140 days in all muscles of T animals compared to C ones (0.01< p <0.05). Collagen concentrations of EDL and RF muscles were also higher (14 to 19%) in T than in C rabbits at 90 and 140 days (0.001 < p < 0.05). In the whole sample, collagen concentration was negatively associated with the procollagen alpha(1)(I) mRNA level in EDL and RF muscles (- 0.49 < r < (- 0.44, p < 0.05), while being positively related to mRNA expression in SMP and Psoas muscles (0.65 < r < 0.85, p < 0.01). It is concluded that jump training clearly restricts the decrease of procollagen (I) mRNA level and probably affects collagen synthesis level. In trained rabbit muscles, the maintenance of a better synthesis level could partly explain the higher collagen concentrations found in EDL and RF at 140 days. Nevertheless, the collagen degradation process seems to play the main role in the increase of total collagen concentration with age in EDL and RF muscles. PMID:15064425

  19. A Myocardial Slice Culture Model Reveals Alpha-1A-Adrenergic Receptor Signaling in the Human Heart

    PubMed Central

    Thomas, R. Croft; Singh, Abhishek; Cowley, Patrick; Myagmar, Bat-Erdene; Montgomery, Megan D.; Swigart, Philip M.; De Marco, Teresa; Baker, Anthony J.; Simpson, Paul C.

    2016-01-01

    Background Translation of preclinical findings could benefit from a simple, reproducible, high throughput human model to study myocardial signaling. Alpha-1A-adrenergic receptors (ARs) are expressed at very low levels in the human heart, and it is unknown if they function. Objectives To develop a high throughput human myocardial slice culture model, and to test the hypothesis that alpha-1A- ARs are functional in the human heart. Methods Cores of LV free wall 8 mm diameter were taken from 52 hearts (18 failing and 34 nonfailing). Slices 250 μm thick were cut with a Krumdieck apparatus and cultured using a rotating incubation unit. Results About 60 slices were cut from each LV core, and a typical study could use 96 slices. Myocyte morphology was maintained, and diffusion into the slice center was rapid. Slice viability was stable for at least 3 days in culture by ATP and MTT assays. The beta-AR agonist isoproterenol stimulated phospholamban phosphorylation, and the alpha-1A-AR agonist A61603 stimulated ERK phosphorylation, with nanomolar EC50 values in slices from both failing and nonfailing hearts. Strips cut from the slices were used to quantify activation of contraction by isoproterenol, A61603, and phenylephrine. The slices supported transduction by adenovirus. Conclusions We have developed a simple, high throughput LV myocardial slice culture model to study signaling in the human heart. This model can be useful for translational studies, and we show for the first time that the alpha-1A-AR is functional in signaling and contraction in the human heart. PMID:27453955

  20. Heterogeneity of alpha1 receptors associated with vascular smooth muscle: evidence from functional and ligand binding studies

    SciTech Connect

    Babich, M.; Pedigo, N.W.; Butler, B.T.; Piascik, M.T.

    1987-08-10

    The nature of the alpha1 receptor associated with rabbit aorta has been examined in functional and receptor binding studies. In isolated aortic rings the dose-response curve for (-)metaraminol was not parallel to that of (-)epinephrine, (-)norepinephrine or (-)phenylephrine. Following inactivation of a portion of the alpha receptors with phenoxybenzamine, the occupancy versus response relationship for metaraminol, in contrast to the other test agonists, was biphasic. In microsomes prepared from aorta, metaraminol bound to two classes of sites labelled by the selective alpha1 antagonist (TH) prazosin. Norepinephrine also bound to two sites on the alpha receptor in all three preparations tested. The Scatchard plot of (TH)prazosin binding to microsomes prepared from frozen aorta was curvilinear. Estimates of the affinities and site densities were 49.6 +/- 15.3 pM and 44.8 +/- 11.8 pmol/gm protein and 1.0 +/- 0.2 nM and 43.8 +/- 17.4 pmol/gm for the high and low affinity sites, respectively. These data are consistent with the idea that there are subtypes of the alpha1 receptor. 33 references, 5 figures.

  1. Effect of aging on alpha-1 adrenergic stimulation of phosphoinositide hydrolysis in various regions of rat brain

    SciTech Connect

    Burnett, D.M.; Bowyer, J.F.; Masserano, J.M.; Zahniser, N.R. )

    1990-12-01

    The effects of aging were examined on the ability of alpha-1 adrenergic receptor agonists to stimulate phosphoinositide hydrolysis in three brain regions. Tissue minces of thalamus, cerebral cortex and hippocampus from 3-, 18- and 28-month-old male Fischer 344 rats were prelabeled with ({sup 3}H)myoinositol. Exposure of these prelabeled minces to phenylephrine and (-)-norepinephrine revealed that accumulation of ({sup 3}H)inositol phosphates was selectively reduced by 20 to 30% in the thalamus and cerebral cortex of the oldest age group. Analysis of concentration-response and competition binding curves indicated that this decrease was due to diminished agonist efficacy rather than diminished receptor affinity. The reduction in responsiveness to phenylephrine and (-)-norepinephrine in the cerebral cortex and the lack of any changes in the hippocampus parallel previously reported changes in the density of alpha-1 adrenergic receptors with aging. These data indicate that the ability of alpha-1 adrenergic receptor agonists to stimulate phosphoinositide hydrolysis is reduced in some, but not all, brain regions of aged Fischer 344 rats.

  2. Characterization of the alpha-1,2-N-acetylglucosaminyltransferase of Neisseria gonorrhoeae, a key control point in lipooligosaccharide biosynthesis.

    PubMed

    Wakarchuk, Warren; Schur, Melissa J; St Michael, Frank; Li, Jinjuan; Eichler, Eva; Whitfield, Dennis

    2004-06-01

    The biosynthesis of the lipooligosaccharide (LOS) in Neisseria meningitidis has a control point that regulates the extension of the alpha-chain on heptose (I) of the LOS. The gene that encodes the protein responsible for this control had been identified elsewhere, but the enzyme encoded by the gene was not characterized. We have now shown that this same control mechanism operates in the related species, Neisseria gonorrhoeae, using a gene knockout and subsequent characterization of the LOS species produced. We also cloned and expressed the enzyme from both of these pathogens. Using a synthetic acceptor substrate, we have shown unequivocally that the enzyme is an alpha-1,2-N-acetylglucosaminyltransferase. Experiments with both the core oligosaccharide and the synthetic acceptors suggests that the addition of the alpha-1,2-N-acetylglucosamine moiety on the heptose (II) residue precedes the addition of the ethanolamine phosphate at the O3 position on this heptose (II), and that in the absence of the alpha-1,2-N-acetylglucosamine moiety leads to the addition of an extra ethanolamine phosphate on the heptose (II) residue. Our data do not support the hypothesis that ethanolamine phosphate at O3 of heptose (II) is added and is then required for the addition of the N-acetylglucosamine at O2 by the LgtK enzyme. This enzyme represents a control point in the biosynthesis of the LOS of this pathogen and is a potential target for therapeutic intervention. PMID:15044393

  3. Polymorphism and structure of the gene coding for the alpha 1 subunit of the Artemia franciscana Na/K-ATPase.

    PubMed Central

    García-Sáez, A; Perona, R; Sastre, L

    1997-01-01

    Genomic clones coding for one of the two identified Artemia franciscana Na/K-ATPase alpha subunits, the alpha 1 subunit, have been isolated. Several overlapping clones were obtained, although their restriction maps showed a large heterogeneity. Sequencing of their exons showed that they differ in up to 3.46% of their nucleotides in translated regions and 8.18% in untranslated regions. Southern blot analysis of DNA purified from different lots of A. franciscana cysts and from isolated individuals suggests that the variation is due to the existence of multiple Na/K-ATPase alpha 1 subunit alleles in A. franciscana. The Na/K-ATPase alpha 1 subunit gene is divided into 15 exons. Ten of the 14 introns are located in identical positions in this gene as in the human Na/K-ATPase alpha 3 subunit gene. Analysis of the 5' flanking region of the gene has allowed identification of the transcription-initiation sites. The adjacent upstream region has been shown to have functional promoter activity in cultured mammalian cells, suggesting the evolutionary conservation of some of the promoter regulatory sequences. PMID:9020888

  4. Noradrenaline acting on alpha1-adrenoceptor mediates REM sleep deprivation-induced increased membrane potential in rat brain synaptosomes.

    PubMed

    Das, Gitanjali; Mallick, Birendra Nath

    2008-01-01

    We hypothesized that one of the functions of REM sleep is to maintain brain excitability and therefore, REM sleep deprivation is likely to modulate neuronal transmembrane potential; however, so far there was no direct evidence to support the claim. In this study a cationic dye, 3,3'-diethylthiacarbocyanine iodide was used to estimate the potential in synaptosomal samples prepared from control and REM sleep deprived rat brains. The activity of Na-K-ATPase that maintains the transmembrane potential was also estimated in the same sample. Further, the roles of noradrenaline and alpha1-adrenoceptor in mediating the responses were studied both in vivo as well as in vitro. Rats were REM sleep deprived for 4 days by the classical flower-pot method; large platform and recovery controls were carried out in addition to free-moving control. The fluorescence intensity increased in samples prepared from REM sleep deprived rat brain as compared to control, which reflected synaptosomal depolarization after deprivation. The Na-K-ATPase activity also increased in the same deprived sample. Furthermore, both the effects were mediated by noradrenaline acting on alpha1-adrenoceptors in the brain. This is the first direct evidence showing that REM sleep deprivation indeed increased neuronal depolarization, which is the likely cause for increased brain excitability, thus supporting our hypothesis and the effect was mediated by noradrenaline acting through the alpha1-adrenoceptor.

  5. Characterization of a cDNA encoding a novel human Golgi alpha 1, 2-mannosidase (IC) involved in N-glycan biosynthesis.

    PubMed

    Tremblay, L O; Herscovics, A

    2000-10-13

    A human cDNA encoding a 70.9-kDa type II membrane protein with sequence similarity to class I alpha1,2-mannosidases was isolated. The enzymatic properties of the novel alpha1,2-mannosidase IC were studied by expressing its catalytic domain in Pichia pastoris as a secreted glycoprotein. alpha1,2-Mannosidase IC sequentially hydrolyzes the alpha1,2-linked mannose residues of [(3)H]mannose-labeled Man(9)GlcNAc to form [(3)H]Man(6)GlcNAc and a small amount of [(3)H]Man(5)GlcNAc. The enzyme requires calcium for activity and is inhibited by both 1-deoxymannojirimycin and kifunensine. The order of mannose removal was determined by separating oligosaccharide isomers formed from pyridylaminated Man(9)GlcNAc(2) by high performance liquid chromatography. The terminal alpha1,2-linked mannose residue from the middle branch is the last mannose removed by the enzyme. This residue is the mannose cleaved from Man(9)GlcNAc(2) by the endoplasmic reticulum alpha1, 2-mannosidase I to form Man(8)GlcNAc(2) isomer B. The order of mannose hydrolysis from either pyridylaminated Man(9)GlcNAc(2) or Man(8)GlcNAc(2) isomer B differs from that previously reported for mammalian Golgi alpha1,2-mannosidases IA and IB. The full-length alpha1,2-mannosidase IC was localized to the Golgi of MDBK and MDCK cells by indirect immunofluorescence. Northern blot analysis showed tissue-specific expression of a major transcript of 3.8 kilobase pairs. The expression pattern is different from that of human Golgi alpha1,2-mannosidases IA and IB. Therefore, the human genome contains at least three differentially regulated Golgi alpha1, 2-mannosidase genes encoding enzymes with similar, but not identical specificities. PMID:10915796

  6. Alpha1-adrenoceptors trigger the snake venom production cycle in secretory cells by activating phosphatidylinositol 4,5-bisphosphate hydrolysis and ERK signaling pathway.

    PubMed

    Kerchove, Celine M; Luna, Milene S A; Zablith, Mariana B; Lazari, Maria F M; Smaili, Soraya S; Yamanouye, Norma

    2008-08-01

    Loss of venom from the venom gland after biting or manual extraction leads to morphological changes in venom secreting cells and the start of a cycle of production of new venom. We have previously shown that stimulation of both alpha- and beta-adrenoceptors in the secretory cells of the venom gland is essential for the onset of the venom production cycle in Bothrops jararaca. We investigated the signaling pathway by which the alpha-adrenoceptor initiates the venom production cycle. Our results show that the alpha(1)-adrenoceptor subtype is present in venom gland of the snake. In quiescent cells, stimulation of alpha(1)-adrenoceptor with phenylephrine increased the total inositol phosphate concentration, and this effect was blocked by the phospholipase C inhibitor U73122. Phenylephrine mobilized Ca(2+) from thapsigargin-sensitive stores and increased protein kinase C activity. In addition, alpha(1)-adrenoceptor stimulation increased the activity of ERK 1/2, partially via protein kinase C. Using RT-PCR approach we obtained a partial sequence of a snake alpha(1)-adrenoceptor (260 bp) with higher identity with alpha(1D) and alpha(1B)-adrenoceptors from different species. These results suggest that alpha(1)-adrenoceptors in the venom secreting cells are probably coupled to a G(q) protein and trigger the venom production cycle by activating the phosphatidylinositol 4,5-bisphosphate and ERK signaling pathway.

  7. Mixture-based combinatorial libraries from small individual peptide libraries: a case study on α1-antitrypsin deficiency.

    PubMed

    Chang, Yi-Pin; Chu, Yen-Ho

    2014-05-16

    The design, synthesis and screening of diversity-oriented peptide libraries using a "libraries from libraries" strategy for the development of inhibitors of α1-antitrypsin deficiency are described. The major buttress of the biochemical approach presented here is the use of well-established solid-phase split-and-mix method for the generation of mixture-based libraries. The combinatorial technique iterative deconvolution was employed for library screening. While molecular diversity is the general consideration of combinatorial libraries, exquisite design through systematic screening of small individual libraries is a prerequisite for effective library screening and can avoid potential problems in some cases. This review will also illustrate how large peptide libraries were designed, as well as how a conformation-sensitive assay was developed based on the mechanism of the conformational disease. Finally, the combinatorially selected peptide inhibitor capable of blocking abnormal protein aggregation will be characterized by biophysical, cellular and computational methods.

  8. A galactosyl(alpha 1-3)mannose epitope on phospholipids of Leishmania mexicana and L. braziliensis is recognized by trypanosomatid-infected human sera.

    PubMed Central

    Avila, J L; Rojas, M

    1990-01-01

    An immunoglobulin M antibody reactive with galactosyl(alpha 1-3)mannose [Gal(alpha 1-3)Man] residues present on phospholipids extracted from Leishmania mexicana and L. braziliensis was found to be present in high titer in the serum of every normal individual studied. Periodate oxidation, acid hydrolysis, or acetylation suppressed immunoreactivity, suggesting that an oligosaccharide chain was responsible for antibody binding. Interaction occurs only with alpha-Gal terminal residues, since treatment of purified glycophospholipids with alpha-galactosidase but not with beta-galactosidase abolished it. Antibody bound to galactosyl(alpha 1-3)galactose-linked synthetic antigens but did not bind to the same residues present in rabbit, rat, and guinea pig erythrocytes or in murine laminin. Antigen-antibody binding was strongly blocked with Gal(alpha 1-3)Man and Gal(beta 1-4)Man. These results plus inhibition studies with several oligosaccharides suggest that they are indeed different from antibodies against the galactosyl(alpha 1-3)galactose residue. Anti-Gal(alpha 1-3)Man antibody values were significantly elevated in 89% of patients with diffuse cutaneous leishmaniasis, 84% of patients with localized cutaneous leishmaniasis, 69% of patients with mucocutaneous leishmaniasis, and 44 and 62% of patients with Trypanosoma cruzi or T. rangeli infection, respectively, but not in patients with 15 other different infectious and inflammatory diseases. Anti-Gal(alpha 1-3)Man antibody readily absorbed to American Leishmania and Trypanosoma culture forms, suggesting a surface membrane localization of reactive epitope. Gal(alpha 1-3)Man-bearing glycophospholipid was easily extracted from American Leishmania promastigotes and T. cruzi trypomastigotes as well as from American Trypanosoma culture forms. The possibility that this antibody arises against parasitic glycophospholipid-linked Gal(alpha 1-3)Man terminal residues is proposed. PMID:1696285

  9. Automated high-content live animal drug screening using C. elegans expressing the aggregation prone serpin α1-antitrypsin Z.

    PubMed

    Gosai, Sager J; Kwak, Joon Hyeok; Luke, Cliff J; Long, Olivia S; King, Dale E; Kovatch, Kevin J; Johnston, Paul A; Shun, Tong Ying; Lazo, John S; Perlmutter, David H; Silverman, Gary A; Pak, Stephen C

    2010-01-01

    The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms. PMID:21103396

  10. Effect of early diabetes on the expression of alpha-1 adrenergic receptors in aorta and carotid arteries of Wistar Kyoto and spontaneously hypertensive rats.

    PubMed

    Edith-Rodriguez, Jessica; Resendiz-Albor, Aldo Arturo; Arciniega-Martinez, Ivonne Maciel; Campos-Rodriguez, Rafael; Hong, Enrique; Huang, Fengyang; Villafaña, Santiago

    2013-01-01

    Hypertension and diabetes have been related to noradrenergic system impairment, especially to the response mediated by alpha-1 receptors. The aim of this work was to investigate possible changes in the expression of alpha-1 adrenergic receptors in aorta and carotid arteries of Wistar Kyoto and spontaneously hypertensive rats after 4 weeks of the onset of diabetes. Our results suggest that early diabetes modifies the expression of alpha-1 adrenergic receptors in aorta and carotid arteries of both WKY and SHR strains in a different way.

  11. The ontogeny of renal alpha 1- and alpha 2-adrenoceptors in the Dahl rat model of experimental hypertension.

    PubMed

    McCaughran, J A; Juno, C J; O'Malley, E; Rosenthal, M

    1986-09-01

    [3H]prazosin (PRAZ) and [3H]rauwolscine (RAUW) were used to examine the ontogeny of renal alpha 1- and alpha 2-adrenoceptors in the inbred Dahl hypertension-sensitive (S/JR) and -resistant (R/JR) rat. PRAZ and RAUW each bound to a single population of non-interacting sites. The binding of each ligand was saturable and reversible. The greatest proliferation of each receptor subtype occurred between 5 and 25 days of age. During this period, a 4 to 5-fold increase in the density of each was observed. Adult levels of each were reached by 50 days of age. The alpha 2-adrenoceptor was the predominant subtype present in renal tissue. However, its ratio to the alpha 1 subtype was influenced by strain and age: the ratio was greatest in the S/JR strain and decreased with age in both strains. The profile of alpha 1-adrenoceptor development was similar in S/JR and R/JR rats. In contrast, the density of alpha 2-adrenoceptors was similar in S/JR and R/JR rats at 5 and 15 days of age but significantly greater in the S/JR rat between 25 and 150 days of age. The elevated density of alpha 2-adrenoceptors could not be explained by strain-related differences in blood pressure or alterations in the affinity of the receptor. The results suggest that a relationship may exist between elevated renal alpha 2-adrenoceptor density and the genetic predisposition to hypertension in the S/JR rat. However, because this relationship is not apparent during the neonatal period of development, the possibility that the elevated density of sites may be secondary to some other event should also be considered.

  12. Alpha 1 beta 1 integrin on neural crest cells recognizes some laminin substrata in a Ca(2+)-independent manner

    PubMed Central

    1992-01-01

    Neural crest cells migrate along pathways containing laminin and other extracellular matrix molecules. In the present study, we functionally and biochemically identify an alpha 1 beta 1 integrin heterodimer which bears the HNK-1 epitope on neural crest cells. Using a quantitative cell adhesion assay, we find that this heterodimer mediates attachment to laminin substrata prepared in the presence of Ca2+. Interestingly, neural crest cells bind to laminin-Ca2+ substrata in the presence or absence of divalent cations in the cell attachment medium. In contrast, the attachment of neural crest cells to laminin substrata prepared in the presence of EDTA, heparin, Mg2+, or Mn2+ requires divalent cations. Interactions with these laminin substrata are mediated by a different integrin heterodimer, since antibodies against beta 1 but not alpha 1 integrins inhibit neural crest cell attachment. Thus, the type of laminin substratum appears to dictate the choice of laminin receptor used by neural crest cells. The laminin conformation is determined by the ratio of laminin to Ca2+, though incorporation of heparin during substratum polymerization alters the conformation even in the presence of Ca2+. Once polymerized, the substratum appears stable, not being altered by soaking in either EDTA or divalent cations. Our findings demonstrate: (a) that the alpha 1 beta 1 integrin can bind to some forms of laminin in the absence of soluble divalent cations; (b) that substratum preparation conditions alter the conformation of laminin such that plating laminin in the presence of Ca2+ and/or heparin modulates its configuration; and (c) that neural crest cells utilize different integrins to recognize different laminin conformations. PMID:1280273

  13. Distribution pattern and functional state of alpha 1-antichymotrypsin in plaques and vascular amyloid in Alzheimer's disease. A immunohistochemical study with monoclonal antibodies against native and inactivated alpha 1-antichymotrypsin.

    PubMed

    Rozemuller, J M; Abbink, J J; Kamp, A M; Stam, F C; Hack, C E; Eikelenboom, P

    1991-01-01

    Monoclonal antibodies (mAbs) were raised against inactivated alpha 1-antichymotrypsin (ACT) to study the presence and functional state of the serine protease inhibitor alpha 1-antichymotrypsin in cerebral amyloid deposits in Alzheimer's disease. A panel of seven different mAbs was obtained; six of them were directed against neoepitopes that are expressed on ACT after interaction with proteases (inactivated ACT) and one mAb was directed against an epitope that is exposed both on native and inactivated ACT. The mAbs against neoepitopes could discriminate native ACT from complexed and inactivated ACT in vitro as shown in binding experiments in the presence of either native or inactivated ACT. With the mAbs against ACT we found that: (a) besides classical congophilic plaques, amorphous noncongophilic beta/A4-positive plaques were stained; (b) amorphous and classical plaques reacted with both types of mAbs against ACT indicating that this ACT was either complexed to a protease or proteolytically inactivated; (c) vascular amyloid was not stained for ACT. The presence of ACT in amorphous and classical plaques and its absence in vascular amyloid may indicate differences in the proteolytic degradation of preamyloid into amyloid fibrils. Our study strongly suggests that ACT is biologically active in amyloid plaques from an early stage.

  14. Determination of the kappa anti-alpha(1,3) dextran immune response difference by A gene(s) in the VKappa-locus of mice

    PubMed Central

    1979-01-01

    Mice lacking the V(alpha(1,3) (h gamma1)-gene do not produce a gamma1 anti-alpha(1,3) dextran response. However, on hyperimmunization some strains mount a kappa-anti-alpha(1,3) dextran response, whereas other remain nonresponder. Responsiveness in dominant. The kappa-anti- alpha(1,3) response difference is linked to the Ly-3 locus on chromosone 6 and is likely the result of a structural Vkappa-gene(s). In conjunction with previous work, three Vkappa-allogroups can now be distinguished. At present, this is the only example of an immune responsiveness difference associated with the Vkappa-locus. PMID:109565

  15. Biochemical properties and subcellular distribution of the BI and rbA isoforms of alpha 1A subunits of brain calcium channels

    PubMed Central

    1996-01-01

    Biochemical properties and subcellular distribution of the class A calcium channel alpha 1 subunits (alpha 1A) from rat and rabbit brain were examined using site-directed anti-peptide antibodies specific for rat rbA (anti-CNA3) and for rabbit BI (anti-NBI-1 and anti-NBI-2) isoforms of alpha 1A. In immunoblotting experiments, anti-CNA3 specifically identifies multiple alpha 1A polypeptides with apparent molecular masses of 210, 190, and 160 kD, and anti-NBI-1 and anti-NBI-2 specifically recognize 190-kD alpha 1A polypeptides in rat brain membrane. In rabbit brain, anti-NBI-1 or anti-NBI-2 specifically detect alpha 1A polypeptides with apparent molecular masses of 220, 200, and 190 kD, while anti-CNA3 specifically recognizes 190-kD alpha 1A polypeptides. These polypeptides evidently represent multiple isoforms of alpha 1A present in both rat and rabbit brain. Anti-CNA3 specifically immunoprecipitates high affinity receptor sites for omega- conotoxin MVIIC (Kd approximately 100 pM), whereas anti-NBI-2 immunoprecipitates two distinct affinity receptor sites for omega- conotoxin MVIIC (Kd approximately 100 pM and approximately 1 microM). Coimmunoprecipitation experiments indicate that alpha 1A subunits recognized by anti-CNA3 and anti-NBI-2 are associated with syntaxin in a stable, SDS-resistant complex and with synaptotagmin. Immunofluorescence studies reveal that calcium channels recognized by anti-NBI-2 are localized predominantly in dendrites and nerve terminals forming synapses on them, while calcium channels recognized by anti- CNA3 are localized more prominently in cell bodies and in nerve terminals. The mossy fiber terminals in hippocampus and the terminals of climbing and parallel fibers in cerebellum are differentially stained by these isoform-specific antibodies. These results indicate that both rbA and BI isoforms of alpha 1A are expressed in rat and rabbit brain and form calcium channels having alpha 1A subunits with distinct molecular mass, pharmacology

  16. A single aromatic amino acid at the carboxyl terminus of Helicobacter pylori {alpha}1,3/4 fucosyltransferase determines substrate specificity.

    PubMed

    Ma, Bing; Lau, Leon H; Palcic, Monica M; Hazes, Bart; Taylor, Diane E

    2005-11-01

    Fucosyltransferases (FucT) from different Helicobacter pylori strains display distinct Type I (Galbeta1,3GlcNAc) or Type II (Galbeta1,4GlcNAc) substrate specificity. FucT from strain UA948 can transfer fucose to the OH-3 of Type II acceptors as well as to the OH-4 of Type I acceptors on the GlcNAc moiety, so it has both alpha1,3 and alpha1,4 activities. In contrast, FucT from strain NCTC11639 has exclusive alpha1,3 activity. Our domain swapping study (Ma, B., Wang, G., Palcic, M. M., Hazes, B., and Taylor, D. E. (2003) J. Biol. Chem. 278, 21893-21900) demonstrated that exchange of the hypervariable loops, (347)DNPFIFC(353) in 11639FucT and (345)CNDAHYSALH(354) in UA948FucT, were sufficient to either confer or abolish alpha1,4 activity. Here we performed alanine scanning site-directed mutagenesis to identify which amino acids within (345)CNDAHYSALH(354) of UA948FucT confer Type I substrate specificity. The Tyr(350) --> Ala mutation dramatically reduced alpha1,4 activity without lowering alpha1,3 activity. None of the other alanine substitutions selectively eliminated alpha1,4 activity. To elucidate how Tyr(350) determines alpha1,4 specificity, mutants Tyr(350) --> Phe, Tyr(350) --> Trp, and Tyr(350) --> Gly were constructed in UA948FucT. These mutations did not decrease alpha1,3 activity but reduced the alpha1,4 activity to 66.9, 55.6, and 3.1% [corrected] of wild type level, respectively. Apparently the aromatic nature, but not the hydroxyl group of Tyr(350), is essential for alpha1,4 activity. Our data demonstrate that a single amino acid (Tyr(350)) in the C-terminal hypervariable region of UA948FucT determines Type I acceptor specificity. Notably, a single aromatic residue (Trp) has also been implicated in controlling Type I acceptor preference for human FucT III, but it is located in an N-terminal hypervariable stem domain.

  17. Sustained augmentation of cardiac alpha1A-adrenergic drive results in pathological remodeling with contractile dysfunction, progressive fibrosis and reactivation of matricellular protein genes.

    PubMed

    Chaulet, H; Lin, F; Guo, J; Owens, W A; Michalicek, J; Kesteven, S H; Guan, Z; Prall, O W; Mearns, B M; Feneley, M P; Steinberg, S F; Graham, R M

    2006-04-01

    We previously reported that transgenic (TG) mice with cardiac-restricted alpha(1A)-adrenergic receptor (alpha(1A)-AR)-overexpression showed enhanced contractility, but no hypertrophy. Since chronic inotropic enhancement may be deleterious, we investigated if long-term, cardiac function and longevity are compromised. alpha(1A)-TG mice, but not their non-TG littermates (NTLs), showed progressive loss of left ventricular (LV) hypercontractility (dP/dt(max): 14,567+/-603 to 11,610+/-915 mmHg/s, P<0.05, A1A1 line: 170-fold overexpression; and 13,625+/-826 to 8322+/-682 mmHg/s, respectively, P<0.05, A1A4 line: 112-fold overexpression, at 2 and 6 months, respectively). Both TG lines developed LV fibrosis, but not LV dilatation or hypertrophy, despite activation of hypertrophic signaling pathways. Microarray and real time RT-PCR analyses revealed activation of matricellular protein genes, including those for thrombospondin 1, connective tissue growth factor and tenascin C, but not transforming growth factor beta1. Life-span was markedly shortened (mean age at death: 155 days, A1A1 line; 224 days, A1A4 line compared with NTLs: >300 days). Telemetric electrocardiography revealed that death in the alpha(1A)-AR TG mice was due to cardiac standstill preceded by a progressive diminution in QRS amplitude, but not by arrhythmias. The QRS changes and sudden death could be mimicked by alpha(1)-AR activation, and reversed preterminally by alpha(1)-AR blockade, suggesting a relationship to stress- or activity-associated catecholamine release. Thus, long-term augmentation of cardiac alpha(1A)-adrenergic drive leads to premature death and progressive LV fibrosis with reactivation of matricellular protein genes. To our knowledge this is the first evidence in vivo for a role of the alpha(1A)-AR in ventricular fibrosis and in pathological cardiac remodeling.

  18. Sympathetic denervation does not alter the density or properties of alpha-1 adrenergic receptors in rat vas deferens

    SciTech Connect

    Abel, P.W.; Johnson, R.D.; Martin, T.J.; Minneman, K.P.

    1985-06-01

    Alpha-1 adrenergic receptors in surgically denervated rat vas deferens were studied using radioligand binding assays of (/sup 125/I) BE 2254 ((/sup 125/I)BE) and contraction measurements. Scatchard analysis of saturation isotherms of specific (/sup 125/I)BE binding showed no change in the affinity or density of binding sites 4, 7 or 14 days after denervation of rat vas deferens. The potency of norepinephrine in inhibiting specific (/sup 125/I)BE binding was also unchanged 7 days after denervation of vas deferens. The potency of phenylephrine in causing contractions in vitro did not change 4, 7 or 14 days after denervation of vas deferens; however, there was a significant increase in the maximum contractile response to phenylephrine at all time points. After partial inactivation of alpha-1 adrenergic receptors in vitro with phenoxybenzamine, there was an equivalent reduction in the number of (/sup 125/I)BE binding sites in the control and 14-day denervated vas deferens. The equilibrium dissociation constants calculated from contractile measurements for norepinephrine were the same in the control and denervated tissues. However, there was a 2.2-fold increase in contractile sensitivity to norepinephrine 14 days after denervation and a 3.6-fold increase in contractile sensitivity to methacholine 7 days after denervation.

  19. Randomized trial to evaluate the immunorestorative properties of synthetic thymosin-alpha 1 in patients with lung cancer

    SciTech Connect

    Schulof, R.S.; Lloyd, M.J.; Cleary, P.A.; Palaszynski, S.R.; Mai, D.A.; Cox, J.W. Jr.; Alabaster, O.; Goldstein, A.L.

    1985-04-01

    A randomized trial was performed in 42 postradiotherapy patients with non-small cell lung cancer to determine whether the administration of synthetic thymosin-alpha 1 by either a loading dose or a twice-weekly schedule could accelerate the reconstitution of thymic dependent immunity. The radiotherapy-induced immunosuppression was characterized by an absolute T cell lymphopenia and by impaired T cell function in lymphoproliferative assays. Placebo-treated patients did not show any improvement in T cell numbers or function over 15 weeks of serial immune monitoring, and exhibited gradual depressions of helper T lymphocyte percentages. Patients treated with thymosin by the loading dose regimen exhibited a normalization of T cell function (p = 0.04), whereas patients treated with the twice-weekly schedule maintained normal helper T cell percentages (p = 0.04). Thymosin treatment was associated with significant improvements in relapse-free and overall survival, which was most pronounced for patients with nonbulky tumors. Thymosin-alpha 1 exhibits schedule-dependent immune restorative and homeostatic properties. Large scale Phase III trials are indicated to definitively establish the impact of thymosin therapy in lung cancer patients treated with radiotherapy.

  20. Cloning of the mouse GABA-benzodiazepine receptor. alpha. 1 subunit in a study of alcohol neurosensitivity

    SciTech Connect

    Keir, W.J.; Deitrich, R.A.; Sikela, J.M. )

    1989-02-09

    The inhibitory action of gamma amino butyric acid (GABA) is mediated by its binding to the benzodiazepine (BDZ) receptor and opening of a chloride channel. This receptor contains a variety of binding sites for several behavorially active drugs. Recent studies with SS and LS mice which were selected for differential neurosensitivity to ethanol, suggest that the GABAergic system plays a role in this differential sensitivity. Thus genes controlling the GABAergic system may also influence the acute hypnotic actions of ethanol. As a fist step towards verifying this hypothesis we have cloned and partially sequenced the mouse GABA-BDZ {alpha}1 subunit cDNA using a 40 bp oligonucleotide derived from the N terminus of a published bovine {alpha} subunit cDNA. A positive clone from a mouse brain cDNA library was identified and contains an insert of approximately 2.5 Kb. Partial sequence analysis indicates that this clone corresponds to the mouse homolog of the {alpha}1 subunit of the GABA-BDZ receptor. This clone is being used as a probe to identify restriction fragment length polymorphisms in several mouse genotypes which differ in their neurosensitivity to ethanol in an attempt to identify molecular genetic changes in the GABA-BDZ receptor that are related to differential ethanol neurosensitivity.

  1. Detection of bovine alpha-S1-casein in term and preterm human colostrum with proteomic techniques.

    PubMed

    Orru, S; Di Nicola, P; Giuliani, F; Fabris, C; Conti, A; Coscia, A; Bertino, E

    2013-01-01

    Due to increased social awareness of allergens and population hyper-sensitization, the reported incidence of allergic reactions to food allergens has increased over the past two decades. Cow's milk proteins (CMPs) are among the most common food allergens. The aim of this study was to use proteomics techniques to investigate cow's milk allergens in both full-term human colostrum and in preterm newborns mothers where both groups showed no prior allergen detection -- in order to understand whether cows milk allergens could be a cause of sensitization established through lactation. The most relevant finding was the detection of the intact bovine alpha-S1-casein in both term and preterm colostrum. Using techniques detailed in this paper and which allowed for direct protein identification, beta-lactoglobulin was not detected in any of the colostrum samples. According to our results, bovine alpha 1 casein is considered a major cow's milk allergen, is readily secreted in human milk, and so could be considered a possible cause of sensitization in exclusively breastfed infants. PMID:23755758

  2. Detection of bovine alpha-S1-casein in term and preterm human colostrum with proteomic techniques.

    PubMed

    Orru, S; Di Nicola, P; Giuliani, F; Fabris, C; Conti, A; Coscia, A; Bertino, E

    2013-01-01

    Due to increased social awareness of allergens and population hyper-sensitization, the reported incidence of allergic reactions to food allergens has increased over the past two decades. Cow's milk proteins (CMPs) are among the most common food allergens. The aim of this study was to use proteomics techniques to investigate cow's milk allergens in both full-term human colostrum and in preterm newborns mothers where both groups showed no prior allergen detection -- in order to understand whether cows milk allergens could be a cause of sensitization established through lactation. The most relevant finding was the detection of the intact bovine alpha-S1-casein in both term and preterm colostrum. Using techniques detailed in this paper and which allowed for direct protein identification, beta-lactoglobulin was not detected in any of the colostrum samples. According to our results, bovine alpha 1 casein is considered a major cow's milk allergen, is readily secreted in human milk, and so could be considered a possible cause of sensitization in exclusively breastfed infants.

  3. Effect of alpha 1-adrenoceptor blockade on maximal VO2 and endurance capacity in well-trained athletic hypertensive men.

    PubMed

    Tomten, S E; Kjeldsen, S E; Nilsson, S; Westheim, A S

    1994-07-01

    The effect of alpha 1-adrenoceptor blockade (doxazosin, 4 mg daily) on maximal VO2 and physical endurance capacity in 16 mildly hypertensive, athletic men was investigated in a randomized, placebo-controlled, double-blind, two-period of 4 weeks, cross-over study. The maximal workload obtained during graded bicycle ergometer exercise and the corresponding maximal VO2 were reduced by 16 +/- 3 W (mean +/- SE), (P = .00003) and 3 +/- 1 mL/(kg.min) (P = .0004), respectively, on doxazosin compared with placebo. The running time on a 5000 m track increased by 43 +/- 12 sec on doxazosin (P = .04). Heart rate was unchanged during the running session. Systolic blood pressure was reduced by 9 +/- 4.1 mm Hg (P = .04) immediately after finishing 5000 m. Six subjects reported side effects from doxazosin (headache, fatigue, and leg pain). Thus, antihypertensive treatment with alpha 1-selective adrenoceptor blockade moderately, but significantly, reduces maximal O2 consumption and high intensity physical endurance capacity in mildly hypertensive athletic men. Significantly reduced systolic blood pressure and unchanged heart rate immediately after running, combined with unchanged heart rate during the race may, however, suggest a safer exercise performance.

  4. Preparation of a composite film electrochemically deposited with chitosan and gold nanoparticles for the determination of alpha-1-fetoprotein.

    PubMed

    Liu, Yaxi; Yuan, Ruo; Chai, Yaqin; Hong, Chenglin; Guan, Shu

    2010-06-01

    A novel amperometric immunosensor based on chitosan-gold nanoparticles (Chit-GNPs) composite film and thionine (Thi) was prepared for the determination of alpha-1-fetoprotein (AFP). The immunosensor was prepared by electro-depositing a Chit-GNPs composite matrix on the surface of the glass carbon electrode, then Thi was immobilized onto the Chit-GNPs film using glutaraldehyde as a cross-linker. Furthermore, the GNPs were chemisorbed onto Thi film for immobilization of alpha-1-fetoprotein antibody. The procedure of the immunosensor was characterized by means of cyclic voltammograms. The performance and influencing factors of the resulting immunosensor were studied in details. Under optimal conditions, the immunosensor was highly sensitive to AFP and the linear range covered from 0.40 to 200.0 ng mL(-1) with a detection limit of 0.24 ng mL(-1) at three times background noise. Moreover, the simple and controllable electro-deposition method overcame the irreproducibility for preparing Chit-based immunosensor systems and the proposed immunosensor displayed a satisfactory reproducibility and stability. PMID:19859743

  5. In vivo binding in rat brain and radiopharmaceutical preparation of radioiodinated HEAT, an alpha-1 adrenoceptor ligand

    SciTech Connect

    Couch, M.W.; Greer, D.M.; Thonoor, C.M.; Williams, C.M.

    1988-03-01

    In vivo binding of (/sup 125/I)-2-(beta-(3-iodo-4-hydroxyphenyl)ethylaminomethyl tetralone) ((/sup 125/I)HEAT) to alpha-1 adrenoceptors in the rat brain was determined over 4 hr. Uptake in the thalamus and frontal cortex was approximately 0.1% injected dose per gram tissue. Thalamus/cerebellum ratios of 10:1 and frontal cortex/cerebellum ratios of 5:1 were found at 4 hr. Pretreatment with prazosin, an alpha-1 antagonist, completely inhibited the accumulation of (/sup 125/I)HEAT in thalamus and frontal cortex; yet uptake of radioactivity was not significantly affected by antagonists and agonists for other receptors classes (propranolol, beta-1; apomorphine, D-1; spiperone, D-2). Binding of (/sup 125/I)HEAT is saturable. At 4 hr, (/sup 125/I)HEAT or (/sup 123/I)HEAT was shown to be the only radioactive material in rat thalamus and frontal cortex. Iodine-123 HEAT and (/sup 125/I)HEAT were synthesized as radiopharmaceuticals within 3 hr in 99% radiochemical purity.

  6. Characterization of the specificities of human blood group H gene-specified alpha 1,2-L-fucosyltransferase toward sulfated/sialylated/fucosylated acceptors: evidence for an inverse relationship between alpha 1,2-L-fucosylation of Gal and alpha 1,6-L-fucosylation of asparagine-linked GlcNAc.

    PubMed

    Chandrasekaran, E V; Jain, R K; Larsen, R D; Wlasichuk, K; Matta, K L

    1996-07-01

    The assembly of complex structures bearing the H determinant was examined by characterizing the specificities of a cloned blood group H gene-specified alpha 1,2-L-fucosyltransferase (FT) toward a variety of sulfated, sialylated, or fucosylated Gal beta 1,3/4GlcNAc beta- or Gal beta 1,3GalNAc alpha-based acceptor structures. (a) As compared to the basic type 2, Gal beta 1,4GlcNAc beta-(K(m) = 1.67 mM), the basic type 1 was 137% active (K(m) = 0.83 mM). (b) On C-6 sulfation of Gal, type 1 became 142.1% active and type 2 became 223.0% active (K(m) = 0.45 mM). (c) On C-6 sulfation of GlcNAc, type 2 showed 33.7% activity. (d) On C-3 or C-4 fucosylation of GlcNAc, both types 1 and 2 lost activity. (e) Type 1 showed 70.8% and 5.8% activity, respectively, on C-6 and C-4 O-methylation of GlcNAc. (f) Type 1 retained 18.8% activity on alpha 2,6-sialylation of GlcNAc. (g) Terminal type 1 or 2 of extended chain had lower activity. (h) With Gal in place of GlcNAc in type 1, the activity became 43.2%. (i) Compounds with terminal alpha 1,3-linked Gal were inactive. (j) Gal beta 1,3GalNAc alpha- (the T-hapten) was approximately 0.4-fold as active as Gal beta 1,4GlcNAc beta-. (k) C-6 sulfation of Gal on the T-hapten did not affect the acceptor activity. (l) C-6 sulfation of GalNAc decreased the activity to 70%, whereas on C-6 sulfation of both Gal and GalNAc the T-hapten lost the acceptor ability. (m) C-6 sialylation of GalNAc also led to inactivity. (n) beta 1,6 branching from GalNAc of the T-hapten by a GlcNAc residue or by units such as Gal beta 1, 4GlcNAc-, Gal beta 1,4(Fuc alpha 1,3)GlcNAc-, or 3-sulfoGal beta 1,4GlcNAc- resulted in 111.9%, 282.8%, 48.3%, and 75.3% activities, respectively. (o) The enhancement of enzyme affinity by a sulfo group on C-6 of Gal was demonstrated by an increase (approximately 5-fold) in the K(m) for Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn in presence of 6-sulfoGal beta 1,- 4GlcNAc beta-O-Me (3.0 mM). (p) Among the two sites in

  7. Characterization of the specificities of human blood group H gene-specified alpha 1,2-L-fucosyltransferase toward sulfated/sialylated/fucosylated acceptors: evidence for an inverse relationship between alpha 1,2-L-fucosylation of Gal and alpha 1,6-L-fucosylation of asparagine-linked GlcNAc.

    PubMed

    Chandrasekaran, E V; Jain, R K; Larsen, R D; Wlasichuk, K; Matta, K L

    1996-07-01

    The assembly of complex structures bearing the H determinant was examined by characterizing the specificities of a cloned blood group H gene-specified alpha 1,2-L-fucosyltransferase (FT) toward a variety of sulfated, sialylated, or fucosylated Gal beta 1,3/4GlcNAc beta- or Gal beta 1,3GalNAc alpha-based acceptor structures. (a) As compared to the basic type 2, Gal beta 1,4GlcNAc beta-(K(m) = 1.67 mM), the basic type 1 was 137% active (K(m) = 0.83 mM). (b) On C-6 sulfation of Gal, type 1 became 142.1% active and type 2 became 223.0% active (K(m) = 0.45 mM). (c) On C-6 sulfation of GlcNAc, type 2 showed 33.7% activity. (d) On C-3 or C-4 fucosylation of GlcNAc, both types 1 and 2 lost activity. (e) Type 1 showed 70.8% and 5.8% activity, respectively, on C-6 and C-4 O-methylation of GlcNAc. (f) Type 1 retained 18.8% activity on alpha 2,6-sialylation of GlcNAc. (g) Terminal type 1 or 2 of extended chain had lower activity. (h) With Gal in place of GlcNAc in type 1, the activity became 43.2%. (i) Compounds with terminal alpha 1,3-linked Gal were inactive. (j) Gal beta 1,3GalNAc alpha- (the T-hapten) was approximately 0.4-fold as active as Gal beta 1,4GlcNAc beta-. (k) C-6 sulfation of Gal on the T-hapten did not affect the acceptor activity. (l) C-6 sulfation of GalNAc decreased the activity to 70%, whereas on C-6 sulfation of both Gal and GalNAc the T-hapten lost the acceptor ability. (m) C-6 sialylation of GalNAc also led to inactivity. (n) beta 1,6 branching from GalNAc of the T-hapten by a GlcNAc residue or by units such as Gal beta 1, 4GlcNAc-, Gal beta 1,4(Fuc alpha 1,3)GlcNAc-, or 3-sulfoGal beta 1,4GlcNAc- resulted in 111.9%, 282.8%, 48.3%, and 75.3% activities, respectively. (o) The enhancement of enzyme affinity by a sulfo group on C-6 of Gal was demonstrated by an increase (approximately 5-fold) in the K(m) for Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn in presence of 6-sulfoGal beta 1,- 4GlcNAc beta-O-Me (3.0 mM). (p) Among the two sites in

  8. Functional interactions of phospholemman (PLM) (FXYD1) with Na+,K+-ATPase. Purification of alpha1/beta1/PLM complexes expressed in Pichia pastoris.

    PubMed

    Lifshitz, Yael; Lindzen, Moshit; Garty, Haim; Karlish, Steven J D

    2006-06-01

    Human FXYD1 (phospholemman, PLM) has been expressed in Pichia pastoris with porcine alpha1/His10-beta1 subunits of Na+,K+-ATPase or alone. Dodecyl-beta-maltoside-soluble complexes of alpha1/beta1/PLM have been purified by metal chelate chromatography, either from membranes co-expressing alpha1,His10-beta1, and PLM or by in vitro reconstitution of PLM with alpha1/His10-beta1 subunits. Comparison of functional properties of purified alpha1/His10-beta1 and alpha1/His10-beta1/PLM complexes show that PLM lowered K0.5 for Na+ ions moderately (approximately 30%) but did not affect the turnover rate or Km of ATP for activating Na+,K+-ATPase activity. PLM also stabilized the alpha1/His10-beta1 complex. In addition, PLM markedly (>3-fold) reduced the K0.5 of Na+ ions for activating Na+-ATPase activity. In membranes co-expressing alpha1/His10-beta1 with PLM the K0.5 of Na+ ions was also reduced, compared with the control, excluding the possibility that detergent or lipid in purified complexes compromise functional interactions. When expressed in HeLa cells with rat alpha1, rat PLM significantly raised the K0.5 of Na+ ions, whereas for a chimeric molecule consisting of transmembranes segments of PLM and extramembrane segments of FXYD4, the K0.5 of Na+ ions was significantly reduced, compared with the control. The opposite functional effects in P. pastoris and HeLa cells are correlated with endogenous phosphorylation of PLM at Ser68 or unphosphorylated PLM, respectively, as detected with antibodies, which recognize PLM phosphorylated at Ser68 (protein kinase A site) or unphosphorylated PLM. We hypothesize that PLM interacts with alpha1/His10-beta1 subunits at multiple locations, the different functional effects depending on the degree of phosphorylation at Ser68. We discuss the role of PLM in regulation of Na+,K+-ATPase in cardiac or skeletal muscle cells.

  9. Mouse alpha1(I)-collagen promoter is the best known promoter to drive efficient Cre recombinase expression in osteoblast.

    PubMed

    Dacquin, Romain; Starbuck, Michael; Schinke, Thorsten; Karsenty, Gérard

    2002-06-01

    Cell- and time-specific gene inactivation should enhance our knowledge of bone biology. Implementation of this technique requires construction of transgenic mouse lines expressing Cre recombinase in osteoblasts, the bone forming cell. We tested several promoter fragments for their ability to drive efficient Cre expression in osteoblasts. In the first mouse transgenic line, the Cre gene was placed under the control of the 2.3-kb proximal fragment of the alpha1(I)-collagen promoter, which is expressed at high levels in osteoblasts throughout their differentiation. Transgenic mice expressing this transgene in bone were bred with the ROSA26 reporter (R26R) strain in which the ROSA26 locus is targeted with a conditional LacZ reporter cassette. In R26R mice, Cre expression and subsequent Cre-mediated recombination lead to expression of the LacZ reporter gene, an event that can be monitored by LacZ staining. LacZ staining was detected in virtually all osteoblasts of alpha1(I)-Cre;R26R mice indicating that homologous recombination occurred in these cells. No other cell type stained blue. In the second line studied, the 1.3-kb fragment of osteocalcin gene 2 (OG2) promoter, which is active in differentiated osteoblasts, was used to drive Cre expression. OG2-Cre mice expressed Cre specifically in bone. However, cross of OG2-Cre mice with R26R mice did not lead to any detectable LacZ staining in osteoblasts. Lastly, we tested a more active artificial promoter derived from the OG2 promoter. The artificial OG2-Cre transgene was expressed by reverse transcriptase-polymerase chain reaction in cartilage and bone samples. After cross of the artificial OG2-Cre mice with R26R mice, we detected a LacZ staining in articular chondrocytes but not in osteoblasts. Our data suggest that the only promoter able to drive Cre expression at a level sufficient to induce recombination in osteoblasts is the alpha1(I)-collagen promoter. PMID:12112477

  10. Nmf11 is a novel ENU-induced mutation in the mouse glycine receptor alpha 1 subunit.

    PubMed

    Traka, Maria; Seburn, Kevin L; Popko, Brian

    2006-09-01

    Nmf11 is an N-ethyl-N-nitrosourea-induced recessive mouse mutation. In this article we show that the mutation is in the gene that encodes the glycine receptor alpha 1 subunit (Glra1). The new Glra1 mutation appears to affect glycine's inhibitory neurotransmission in the central nervous system (CNS) of the nmf11 homozygotes, which suffer from a severe startle disease-related phenotype and die by postnatal day 21. The nmf11 mutation involves a C-to-A transition of nucleotide 518, which results in the N46K substitution in the long extracellular NH(2) terminal or ligand-binding domain of the GLRA1 mature protein. The mutation does not result in reduced expression of GLRA1 at the mRNA or protein levels and the mutant glycine receptor localizes properly in synaptic sites of nmf11 homozygotes. PMID:16964444

  11. Lethal osteogenesis imperfecta congenita and a 300 base pair gene deletion for an alpha 1(I)-like collagen.

    PubMed Central

    Pope, F M; Cheah, K S; Nicholls, A C; Price, A B; Grosveld, F G

    1984-01-01

    Broad boned lethal osteogenesis imperfecta is a severely crippling disease of unknown cause. By means of recombinant DNA technology a 300 base pair deletion in an alpha 1(I)-like collagen gene was detected in six patients and four complete parent-child groups including patients with this disease. One from each set of the patients' clinically unaffected parents also carried the deletion, implying that affected patients were genetic compounds. The study suggests that prenatal diagnosis should be possible with 100% accuracy in subjects without the deletion and with 50% accuracy in those who possess it (who would be either heterozygous--normal, or affected with the disease). Images FIG 1 FIG 2 FIG 3 FIG 4 PMID:6419953

  12. Different affinity states of alpha-1 adrenergic receptors defined by agonists and antagonists in bovine aorta plasma membranes

    SciTech Connect

    Jagadeesh, G.; Deth, R.C.

    1987-11-01

    Evidence for a nonlinear relationship between alpha-1 adrenergic receptor occupancy and tissue responses, together with the finding of different affinity states for agonist binding, has raised the possibility of functional heterogeneity of alpha-1 adrenergic receptors. We have conducted studies to examine: 1) binding characteristics of (/sup 3/H)prazosin, 2) competition of antagonists at these sites and 3) different affinity states of the receptor for agonists and modulation of these states by 5'-guanylylimidodiphosphate (Gpp(NH)p). A plasma membrane-enriched vesicular fraction (F2; 15%/33% sucrose interphase) was prepared from the muscular medial layer of bovine thoracic aorta. (/sup 3/H)Prazosin binding was characterized by a monophasic saturation isotherm (KD = 0.116 nM, Bmax = 112 fmol/mg of protein). Antagonist displacement studies yielded a relative potency order of prazosin greater than or equal to WB4104 much greater than phentolamine greater than corynanthine greater than yohimbine greater than or equal to idazoxan greater than rauwolscine. Competition curves for unlabeled prazosin, WB4101 (2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4 benzodioxane) and phentolamine were shallow and were best modeled to two binding sites with picomolar and nanomolar KD values. Gpp(NH)p was without effect on antagonist affinity. Agonist (epinephrine, norepinephrine and phenylephrine) competition with (/sup 3/H)prazosin binding was biphasic with pseudo-Hill slopes less than 1.0. Binding was best described by a two-site model in which the average contribution of high affinity sites was 23% of total binding. KD values for the high affinity site ranged from 2.9 to 18 nM, and 3.9 to 5.0 microM for the low affinity site.

  13. T-cell abnormalities after mediastinal irradiation for lung cancer. The in vitro influence of synthetic thymosin alpha-1

    SciTech Connect

    Schulof, R.S.; Chorba, T.L.; Cleary, P.A.; Palaszynski, S.R.; Alabaster, O.; Goldstein, A.L.

    1985-03-01

    The effects of mediastinal irradiation (RT) on the numbers and functions of purified peripheral blood T-lymphocytes from patients with locally advanced non-small cell lung cancer were evaluated. The patients were candidates for a randomized trial to evaluate the immunorestorative properties of synthetic thymosin alpha-1. Twenty-one patients studied before RT did not exhibit any significant difference in T-cell numbers or function compared to age-matched healthy subjects. However, 41 patients studied within 1 week after completing RT exhibited significant depressions of E-rosette-forming cells at 4 degrees C (E4 degrees-RFC)/mm3, E-rosette-forming cells at 29 degrees C (E29 degrees-RFC)/mm3, OKT3/mm3, OKT4/mm3, and OKT8/mm3 (P . 0.0001); total T-cell percentages (%OKT3, P . 0.01); and T-cell proliferative responses in mixed lymphocyte cultures (MLR) (P . 0.01) and to the mitogen phytohemagglutinin under suboptimal conditions (P less than or equal to 0.03). Nine patients studied before and after RT showed a significant increase in OKT4/OKT8 (P . 0.01) following RT. A short-term in vitro incubation with thymosin alpha-1 could enhance MLR of T-cells in 12 of 27 patients with post-RT abnormalities. In 13 patients who were treated with placebo, the RT-induced depression of T-cell numbers and function persisted for at least 3 to 4 months. In addition, in 12 patients progressive decreases developed in %E4 degrees-RFC, %OKT3, %OKT4, and OKT4/OKT8, which always preceded clinical relapse.

  14. Molecular mechanisms of benzodiazepine-induced down-regulation of GABAA receptor alpha 1 subunit protein in rat cerebellar granule cells.

    PubMed Central

    Brown, M. J.; Bristow, D. R.

    1996-01-01

    1. Chronic benzodiazepine treatment of rat cerebellar granule cells induced a transient down-regulation of the gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit protein, that was dose-dependent (1 nM-1 microM) and prevented by the benzodiazepine antagonist flumazenil (1 microM). After 2 days of treatment with 1 microM flunitrazepam the alpha 1 subunit protein was reduced by 41% compared to untreated cells, which returned to, and remained at, control cell levels from 4-12 days of treatment. Chronic flunitrazepam treatment did not significantly alter the GABAA receptor alpha 6 subunit protein over the 2-12 day period. 2. GABA treatment for 2 days down-regulates the alpha 1 subunit protein in a dose-dependent (10 microM-1 mM) manner that was prevented by the selective GABAA receptor antagonist bicuculline (10 microM). At 10 microM and 1 mM GABA the reduction in alpha 1 subunit expression compared to controls was 31% and 66%, respectively. 3. The flunitrazepam-induced decrease in alpha 1 subunit protein is independent of GABA, which suggests that it involves a mechanism distinct from the GABA-dependent action of benzodiazepines on GABAA receptor channel activity. 4. Simultaneous treatment with flunitrazepam and GABA did not produce an additive down-regulation of alpha 1 subunit protein, but produced an effect of the same magnitude as that of flunitrazepam alone. This down-regulation induced by the combination of flunitrazepam and GABA was inhibited by flumazenil (78%), but unaffected by bicuculline. 5. The flunitrazepam-induced down-regulation of alpha 1 subunit protein at 2 days was completely reversed by the protein kinase inhibitor staurosporine (0.3 microM). 6. This study has shown that both flunitrazepam and GABA treatment, via their respective binding sites, caused a reduction in the expression of the GABAA receptor alpha 1 subunit protein; an effect mediated through the same neurochemical mechanism. The results also imply that the benzodiazepine effect

  15. Centrosymmetric bilayers in the 0.75 A resolution structure of a designed alpha-helical peptide, D,L-Alpha-1.

    PubMed Central

    Patterson, W. R.; Anderson, D. H.; DeGrado, W. F.; Cascio, D.; Eisenberg, D.

    1999-01-01

    We report the 0.75 A crystal structure of a racemic mixture of the 12-residue designed peptide "Alpha-1" (Acetyl-ELLKKLLEELKG), the L-enantiomer of which is described in the accompanying paper. Equivalent solutions of the centrosymmetric bilayers were determined by two direct phasing programs in space groups P1 and P1bar. The unit cell contains two L-alpha-helices and two D-alpha-helices. The columnar-sheet bilayer motif seen in L-Alpha-1 is maintained in the D,L-Alpha-1 structure except that each sheet of head-to-tail helices is composed of one enantiomer and is related to its neighboring sheets by inversion symmetry. Comparison to the L-Alpha-1 structure provides further insight into peptide design. The high resolution and small asymmetric unit allowed building an intricate model (R = 13.1%, Rfree = 14.5%) that incorporates much of the discrete disorder of peptide and solvent. Ethanolamine and 2-methyl-2,4-pentanediol (MPD) molecules bind near helix termini. Rigid body analysis identifies sites of restricted displacements and torsions. Side-chain discrete disorder propagates into the backbone of one helix but not the other. Although no side chain in Alpha-1 is rigid, the environments in the crystal restrict some of them to no or only one active torsion. PMID:10422829

  16. Toxin a from Clostridium difficile binds to rabbit erythrocyte glycolipids with therminal Gal. cap alpha. 1-3Gal. beta. 1-4GlcNaC sequences

    SciTech Connect

    Clark, G.F.; Krivan, H.; Wilkins, T.; Smith, D.F.

    1987-05-01

    Toxin A is one of two clostridial toxins implicated as the causative agent of pseudomembranous colitis in patients undergoing postoperative antibiotic therapy. Evidence that the carbohydrate binding determinant for this toxin is a glycoconjugate(s) with non-reducing Gal..cap alpha..1-3Gal..beta..1-4GlcNAc has recently been reported. Specific agglutination of rabbit erythrocytes by Toxin A is inhibited by bovine thyroglobulin and prevented by pretreatment of cells with ..cap alpha..-galactosidase. Total lipid extracts from rabbit erythrocytes were subjected to thin layer chromatography and the chromatogram overlaid with purified /sup 125/I-labeled Toxin A. Two major and several minor toxin-binding glycolipids were detected following autoradiography. The major toxin-binding glycolipids were identified as pentasaccharide- and decasaccharide-ceramides expressing terminal Gal..cap alpha..1-3Gal..beta..1-4GlcNAc sequences. Treatment of the toxin-binding glycolipids with ..cap alpha..-galactosidase abolished binding. Forsmann glycolipid, globoside, Gal..cap alpha..1-4 Gal..beta..1-4Glc-cer, and Gal..cap alpha..1-3Gal..beta..1-4Glc-cer did not bind the toxin. These observations are consistent with the proposed carbohydrate specificity of the toxin for the non-reducing terminal sequence, Gal..cap alpha..1-3Gal..beta..1-4GlcNAc.

  17. Imbalance between Neutrophil Elastase and its Inhibitor α1-Antitrypsin in Obesity Alters Insulin Sensitivity, Inflammation, and Energy Expenditure

    PubMed Central

    Mansuy-Aubert, Virginie; Zhou, Qiong L.; Xie, Xiangyang; Gong, Zhenwei; Huang, Jun-Yuan; Khan, Abdul R.; Aubert, Gregory; Candelaria, Karla; Thomas, Shantele; Shin, Dong-Ju; Booth, Sarah; Baig, Shahid M.; Bilal, Ahmed; Hwang, Daehee; Zhang, Hui; Lovell-Badge, Robin; Smith, Steven R.; Awan, Fazli R.; Jiang, Zhen Y.

    2013-01-01

    SUMMARY The molecular mechanisms involved in the development of obesity and related complications remain unclear. Here, we report that obese mice and human subjects have increased activity of neutrophil elastase (NE) and decreased serum levels of the NE inhibitor, α1-antitrypsin (A1AT, SerpinA1). NE null (Ela2−/−) mice and A1AT transgenic mice were resistant to high-fat diet (HFD)-induced bodyweight gain, insulin resistance, inflammation and fatty liver. NE inhibitor GW311616A reversed insulin resistance and bodyweight gain in HFD-fed mice. Compared with wild-type mice, Ela2−/− mice augmented circulating high molecular weight (HMW) adiponectin levels, phosphorylation of AMP-activated protein kinase (AMPK) and fatty acid oxidation (FAO) in the liver and brown adipose tissue (BAT), and uncoupling protein (UCP1) levels in the BAT. These data suggest that the A1AT-NE system regulates AMPK signaling, FAO and energy expenditure. The imbalance between A1AT and NE contributes to the development of obesity and related inflammation, insulin resistance and liver steatosis. PMID:23562077

  18. A C. elegans model of human α1-antitrypsin deficiency links components of the RNAi pathway to misfolded protein turnover

    PubMed Central

    Long, Olivia S.; Benson, Joshua A.; Kwak, Joon Hyeok; Luke, Cliff J.; Gosai, Sager J.; O'Reilly, Linda P.; Wang, Yan; Li, Jie; Vetica, Anne C.; Miedel, Mark T.; Stolz, Donna B.; Watkins, Simon C.; Züchner, Stephan; Perlmutter, David H.; Silverman, Gary A.; Pak, Stephen C.

    2014-01-01

    The accumulation of serpin oligomers and polymers within the endoplasmic reticulum (ER) causes cellular injury in patients with the classical form α1-antitrypsin deficiency (ATD). To better understand the cellular and molecular genetic aspects of this disorder, we generated transgenic C. elegans strains expressing either the wild-type (ATM) or Z mutant form (ATZ) of the human serpin fused to GFP. Animals secreted ATM, but retained polymerized ATZ within dilated ER cisternae. These latter animals also showed slow growth, smaller brood sizes and decreased longevity; phenotypes observed in ATD patients or transgenic mouse lines expressing ATZ. Similar to mammalian models, ATZ was disposed of by autophagy and ER-associated degradation pathways. Mutant strains defective in insulin signaling (daf-2) also showed a marked decrease in ATZ accumulation. Enhanced ATZ turnover was associated with the activity of two proteins central to systemic/exogenous (exo)-RNAi pathway: the dsRNA importer, SID-1 and the argonaute, RDE-1. Animals with enhanced exo-RNAi activity (rrf-3 mutant) phenocopied the insulin signaling mutants and also showed increased ATZ turnover. Taken together, these studies allude to the existence of a novel proteostasis pathway that mechanistically links misfolded protein turnover to components of the systemic RNAi machinery. PMID:24838286

  19. The effects of weight gain after smoking cessation on atherogenic α1-antitrypsin-low-density lipoprotein.

    PubMed

    Komiyama, Maki; Wada, Hiromichi; Ura, Shuichi; Yamakage, Hajime; Satoh-Asahara, Noriko; Shimada, Sayaka; Akao, Masaharu; Koyama, Hiroshi; Kono, Koichi; Shimatsu, Akira; Takahashi, Yuko; Hasegawa, Koji

    2015-11-01

    Although cardiovascular risks decrease after quitting smoking, body weight often increases in the early period after smoking cessation. We have previously reported that the serum level of the α1-antitrypsin-low-density lipoprotein complex (AT-LDL)-an oxidatively modified low-density lipoprotein that accelerates atherosclerosis-is high in current smokers, and that the level rapidly decreases after smoking cessation. However, the effects of weight gain after smoking cessation on this cardiovascular marker are unknown. In 183 outpatients (134 males, 49 females) who had successfully quit smoking, serum AT-LDL levels were measured using an enzyme-linked immunosorbent assay. For all persons who had successfully quit smoking, body mass index (BMI) significantly increased 12 weeks after the first examination (p < 0.01). Among patients with a BMI increase smaller than the median, a significant decrease (p < 0.01) in serum AT-LDL values was found, but no significant changes in serum AT-LDL values were found in patients with a BMI increase greater than the median. The findings suggest that the decrease in serum AT-LDL levels after quitting smoking is influenced by weight gain after smoking cessation.

  20. Conformational properties of the disease-causing Z variant of α1-antitrypsin revealed by theory and experiment.

    PubMed

    Kass, Itamar; Knaupp, Anja S; Bottomley, Stephen P; Buckle, Ashley M

    2012-06-20

    The human serine protease inhibitor (serpin) α-1 antitrypsin (α1-AT) protects tissues from proteases of inflammatory cells. The most common disease-causing mutation in α1-AT is the Z-mutation (E342K) that results in an increased propensity of α1-AT to polymerize in the ER of hepatocytes, leading to a lack of secretion into the circulation. The structural consequences of this mutation, however, remain elusive. We report a comparative molecular dynamics investigation of the native states of wild-type and Z α1-AT, revealing a striking contrast between their structures and dynamics in the breach region at the top of β-sheet A, which is closed in the wild-type simulations but open in the Z form. Our findings are consistent with experimental observations, notably the increased solvent exposure of buried residues in the breach region in Z, as well as polymerization via domain swapping, whereby the reactive center loop is rapidly inserted into an open A-sheet before proper folding of the C-terminal β-strands, allowing C-terminal domain swapping with a neighboring molecule. Taken together, our experimental and simulation data imply that mutations at residue 342 that either stabilize an open form of the top of β-sheet A or increase the local flexibility in this region, may favor polymerization and hence aggregation.

  1. Alpha 1-adrenoceptor blocking activities of bevantolol hydrochloride(NC-1400) and labetalol in rat isolated thoracic aorta--do they distinguish between subtypes?

    PubMed

    Shiraishi, K; Moriya, M; Miyake, N; Takayanagi, I

    1992-09-01

    1. alpha 1-adrenoceptor blocking activity of bevantolol(NC-1400) was tested in the rat thoracic aorta, comparing with that of labetalol. 2. In alpha 1-adrenoceptor blocking activity, bevantolol(NC-1400) was 1/20th as potent as labetalol. 3. The pA2 values of bevantolol(NC-1400) and labetalol estimated in the rat thoracic aorta were compared with those in the rabbit thoracic aorta, which were reported by Takayanagi et al. [Takayanagi I., Kizawa Y., Iwasaki S. and Nakagoshi A. (1987) Gen. Pharmac. 18, 87-89]. 4. The pA2 values of bevantolol(NC-1400) and labetalol were approximately 1 order of magnitude higher in rat aorta than in rabbit aorta, suggesting that both the drugs distinguish between subtypes of alpha 1-adrenoceptors.

  2. Toxin A from Clostridium difficile binds to rabbit erythrocyte glycolipids with terminal Gal alpha 1-3Gal beta 1-4GlcNAc sequences

    SciTech Connect

    Clark, G.F.; Krivan, H.C.; Wilkins, T.D.; Smith, D.F.

    1987-08-15

    The binding of Toxin A isolated from Clostridium difficile to rabbit erythrocyte glycolipids has been studied. Total lipid extracts from rabbit erythrocytes were subjected to thin-layer chromatography and toxin-binding glycolipids detected by using /sup 125/I-labeled Toxin A in a direct binding overlay technique. Two major and several minor toxin-binding glycolipids were detected in rabbit erythrocytes by this method. The results of structural analyses of the major toxin-binding glycolipids were consistent with a pentasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) and a branched decasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) previously identified as the two most abundant glycolipids in rabbit erythrocytes. /sup 125/I-Toxin A binding to these glycolipids could be inhibited by bovine thyroglobulin, monospecific antiserum to the toxin, or by treatment of the glycolipids with alpha-galactosidase. The absence of toxin interaction with isoglobotriaosylceramide (Gal alpha 1-3Gal beta 1-4Glc-Cer) isolated from canine intestine suggested that the GlcNAc residue present in the terminal Gal alpha 1-3Gal beta 1-4GLcNAc sequence common to all known toxin binding glycoconjugates is required for carbohydrate-specific recognition by Toxin A. These observations are consistent with the proposed carbohydrate binding specificity of Toxin A for the nonreducing terminal sequence, Gal alpha 1-3Gal beta 1-4GlcNAc.

  3. Interactions of purified transcription factors: binding of yeast MAT alpha 1 and PRTF to cell type-specific, upstream activating sequences.

    PubMed Central

    Tan, S; Ammerer, G; Richmond, T J

    1988-01-01

    Pheromone receptor transcription factor (PRTF) and MAT alpha 1 are protein transcription factors that are involved in the regulation of the alpha-specific genes in Saccharomyces cerevisiae. We have expressed MAT alpha 1 as a fusion protein in Escherichia coli and purified it from inclusion bodies in milligram quantities. The MAT alpha 1 protein was obtained after specific cleavage of the fusion protein. Quantitative band shift electrophoresis was used to determine the equilibrium dissociation constants that describe the multicomponent binding equilibrium between the PRTF and MAT alpha 1 proteins, and alpha-specific STE3 upstream activating sequence (UAS) DNA. The dissociation constant for the complex of PRTF and the a-specific UAS of STE2 was also measured and found to be 5.9 X 10(-11) M, only three times less than that for the PRTF-STE3 UAS complex. Analyses of these complexes by DNase I footprinting demonstrate that the PRTF binding site is confined to the palindromic P-box sequence in the case of the STE3 UAS, but extends symmetrically from this central region to cover 28 bp for the STE2 UAS. When MAT alpha 1 is bound to the PRTF-STE3 complex, the region of DNA protected is enlarged to that seen for the PRTF-STE2 complex. Our results using these two purified factors in vitro suggest that PRTF has nearly the same affinity for a- and alpha-specific UAS elements and that transcriptional activation requires a particular conformational state for the PRTF-DNA complex which occurs in the PRTF-STE2 and MAT alpha 1-PRTF-STE3 complexes, but not in the PRTF-STE3 complex. Images PMID:2854061

  4. Examination by radioligand binding of the alpha1 adrenoceptors in the mesenteric arterial vasculature during the development of salt-sensitive hypertension.

    PubMed

    Caveney, S W; Taylor, D A; Fleming, W W

    1997-09-01

    Previous experiments have suggested that the vascular smooth muscle of Dahl salt-sensitive (DS) rats may possess a difference in the alpha1-adrenoceptor population or its transduction processes compared to Dahl salt-resistant (DR) rats. The purpose of the current research is to study the role of alpha1-adrenoceptors in the specific supersensitivity to norepinephrine (NE) seen prior to and early in the development of hypertension in the DS rat. Experiments in isolated perfused superior mesenteric arterial vasculature from DS rats chronically fed a high (7%) salt diet for 5 days or 3 weeks, in the absence or presence of an elevation in systolic blood pressure, respectively, demonstrated a specific supersensitivity to NE relative to DR rats. The enhanced responsiveness was specific to NE after 5 days of high salt since no differences in sensitivity of these preparations was observed to either KCl or 5-HT. A small but significant elevation in sensitivity to KCl following 3 weeks of treatment suggests that multiple factors may contribute to tissue responsiveness at this time. Radioligand binding experiments were performed using [125I]-HEAT to study the alpha1-adrenoceptor population and its subtypes. Saturation experiments using membranes prepared from the superior mesenteric arterial vasculature or mesenteric arterial branches showed no significant differences in overall alpha1-adrenoceptor population between DS and DR rats fed a high-salt diet for 5 days or 3 weeks. Competition experiments using membranes prepared from the superior mesenteric arterial branches in the presence of the alpha1A-subtype selective antagonist 5-methylurapidil showed two binding sites (high and low affinity) in these resistance vessels but no significant differences in nature or ratio of these sites between the DS and DR groups. These results suggest that changes in the alpha1-adrenoceptor population are not responsible for the specific supersensitivity to NE, which may be an early event in

  5. Cathepsin G and alpha 1-antichymotrypsin in the local host reaction to loosening of total hip prostheses.

    PubMed

    Takagi, M; Konttinen, Y T; Santavirta, S; Kangaspunta, P; Suda, A; Rokkanen, P

    1995-01-01

    The tissue localization and content of the proteolytic enzyme cathepsin G and its inhibitor alpha 1-antichymotrypsin were studied in the local host reaction to loosening of total hip-replacement prostheses in eleven patients and were compared with those in samples of non-inflammatory tissue from the synovial capsule obtained during arthroscopies of the knee. Immunostaining demonstrated cellular localization of cathepsin G in 71 per cent of monocyte or macrophage-like cells and in 46 per cent of fibroblast-like cells in the samples of interface tissue between the bone and the loose acetabular component obtained at the time of the total hip replacements, and in 59 and 42 per cent, respectively, in the samples of pseudocapsular tissue obtained at the same time, whereas the synovial lining cells in the samples of non-inflammatory tissue from the synovial capsule revealed only a slight immunoreactivity to cathepsin G. Cathepsin-G activity was also measured with synthetic succinyl-alanine-alanine-proline-phenylalanine-paranitroanilide as a substrate, the degradation of which was monitored spectrophotometrically. In accordance with results from immunohistochemical studies, cathepsin-G activity was found in the samples of interface tissue (31.6 international units per liter) and the samples of pseudocapsular tissue (15.5 international units per liter) obtained during the total hip replacements, whereas the level of cathepsin-G was low in the samples of non-inflammatory synovial capsular tissue (2.5 international units per liter). Cathepsin-G activity in the samples of pseudosynovial fluid obtained at the time of the total hip replacements was low (2.4 international units per liter), although immunoblot analysis showed marked immunoreactive cathepsin G in the samples of pseudosynovial fluid. This low activity of cathepsin G might be explained by the presence of alpha 1-antichymotrypsin, which was detected by laser nephlometric immunoassay and immunoblot analysis. These

  6. Thymosin alpha 1 antagonizes dexamethasone and CD3-induced apoptosis of CD4+ CD8+ thymocytes through the activation of cAMP and protein kinase C dependent second messenger pathways.

    PubMed

    Baumann, C A; Badamchian, M; Goldstein, A L

    1997-03-01

    It is well established that glucocorticoid hormones and anti-CD3 monoclonal antibodies induce apoptosis in immature developing thymocytes. This process can be modulated by soluble factors, anti-oxidants and adhesion receptors. Previously we have demonstrated that thymosin alpha 1 (T alpha 1), a 28-amino acid thymic peptide hormone, is a dose and time dependent antagonist of dexamethasone (DEX) and CD# induced DNA fragmentation of murine thymocytes in vitro. To further investigate the mechanism of T alpha 1 action we determined a T alpha 1 sensitive thymocyte population and examined some of the molecular events associated with T alpha 1 anti-apoptotic activity. Phenotypic analysis of the sub-populations of thymocytes, based on CD4 and CD8 expression, revealed that T alpha 1 exerts its effect on CD4+ CD8+ immature thymocytes. T alpha 1 treatment of thymocytes delays the production of free radicals and the subsequent consumption of glutathione, that is observed during both DEX and CD3 induced apoptosis. We further demonstrate that T alpha 1 stimulates the production of cAMP and activates PKC in thymocytes. These data suggest that T alpha 1 exerts an influence on the development of a population of immature T-cells in the thymus by effecting the sensitivity of thymocytes to apoptosis during the pre-selection stages of thymic development. Our studies also suggest that the mechanism of T alpha 1 action involves the induction of both cAMP and PKC dependent second messenger pathways.

  7. Intravenous administration of alpha-1-proteinase inhibitor in patients of PiZ and PiM phenotype. Preliminary report

    SciTech Connect

    Moser, K.M.; Smith, R.M.; Spragg, R.G.; Tisi, G.M.

    1988-06-24

    Nine patients with moderate pulmonary emphysema, six of PiZ phenotype and three of PiM phenotype, have received a single intravenous infusion of alpha-1-proteinase inhibitor (human) (A1PI), in a dose of 60 mg/kg over a 30-minute period. They also received a tracer dose (300 microCi) of /sup 131/I-labeled A1PI. No active or passive immunization against hepatitis was given. No acute toxicity was observed. Compared with baseline data, significant elevations of serum A1PI (measured both antigenically and as anti-elastase activity) occurred, with a serum half-life approximating 110 hours. Bronchoalveolar lavage fluid, obtained 48 hours after infusion, reflected a significant increase in A1PI concentration versus baseline bronchoalveolar lavage fluid values. Serial gamma camera images of the lungs confirmed persistence of enhanced lung radioactivity for several days. Urinary desmosine excretion did not change following A1PI infusion. During the period of follow-up thus far, no patient has had chronic toxicity, results of liver function tests have been stable, and there has been no development of hepatitis B antigen or antibodies to hepatitis B surface or core antigens.

  8. Subcellular site of synthesis of the N-acetylgalactosamine (alpha 1-0) serine (or threonine) linkage in rat liver

    SciTech Connect

    Abeijon, C.; Hirschberg, C.B.

    1987-03-25

    We have studied the subcellular site of synthesis of the GalNAc(alpha-1-0) Ser/Thr linkage in rat liver. The specific and total activities of polypeptide:N-acetylgalactosaminyltransferase (using apomucin as exogenous acceptor) were highly enriched in membrane fractions derived from the Golgi apparatus; virtually no activity was detected in membranes from the rough and smooth endoplasmic reticulum. Vesicles of the above organelles (which were sealed and of the same membrane topographical orientation as in vivo) were able to translocate UDP-GalNAc into their lumen in an assay in vitro; the initial translocation rate into Golgi vesicles was 4-6-fold higher than that into vesicles from the rough and smooth endoplasmic reticulum. Translocation of UDP-GalNAc into Golgi vesicles was temperature dependent and saturable with an apparent Km of 8-10 microM. UDP-GalNAc labeled with different radioisotopes in the uridine and sugar was used to determine that the intact sugar nucleotide was being translocated in a reaction coupled to the exit of luminal UMP. Following translocation of UDP-GalNAc, transfer of GalNAc into endogenous macromolecular acceptors was detected in Golgi vesicles and not in those from the rough and smooth endoplasmic reticulum. The above results together with previous studies on the O-xylosylation of the linkage region of proteoglycans strongly suggest that, in rat liver, the bulk of O-glycosylation reactions occur in the Golgi apparatus.

  9. Ape1/Ref-1 induces glial cell-derived neurotropic factor (GDNF) responsiveness by upregulating GDNF receptor alpha1 expression.

    PubMed

    Kim, Mi-Hwa; Kim, Hong-Beum; Acharya, Samudra; Sohn, Hong-Moon; Jun, Jae Yeoul; Chang, In-Youb; You, Ho Jin

    2009-04-01

    Apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) dysregulation has been identified in several human tumors and in patients with a variety of neurodegenerative diseases. However, the function of Ape1/Ref-1 is unclear. We show here that Ape1/Ref-1 increases the expression of glial cell-derived neurotropic factor (GDNF) receptor alpha1 (GFRalpha1), a key receptor for GDNF. Expression of Ape1/Ref-1 led to an increase in the GDNF responsiveness in human fibroblast. Ape1/Ref-1 induced GFRalpha1 transcription through enhanced binding of NF-kappaB complexes to the GFRalpha1 promoter. GFRalpha1 levels correlate proportionally with Ape1/Ref-1 in cancer cells. The knockdown of endogenous Ape1/Ref-1 in pancreatic cancer cells markedly suppressed GFRalpha1 expression and invasion in response to GNDF, while overexpression of GFRalpha1 restored invasion. In neuronal cells, the Ape1/Ref-1-mediated increase in GDNF responsiveness not only stimulated neurite outgrowth but also protected the cells from beta-amyloid peptide and oxidative stress. Our results show that Ape1/Ref-1 is a novel physiological regulator of GDNF responsiveness, and they also suggest that Ape1/Ref-1-induced GFRalpha1 expression may play important roles in pancreatic cancer progression and neuronal cell survival.

  10. Effects of alpha1-adrenoceptor antagonist (tamsulosin) on incident of ejaculation and semen quality in the goat.

    PubMed

    Kimsakulvech, S; Suttiyotin, P; Pinyopummin, A

    2015-04-01

    Male temporary contraception is occasionally required in some animals. Alpha1-adrenoceptor antagonist (tamsulosin) can cause ejaculation disorder. Two sets of Latin square were applied to six male goats to received either normal saline, dimethylsulphoxide or tamsulosin (179.8 nmol kg(-1) ) at 1-week interval. Semen collection and libido scoring were undertaken at 3, 6 and 24 h post-injection. For ejaculated semen, its quality was evaluated. Physiological measurements including body temperature, respiration and heart rates were measured before injection and at 30 min before semen collection. The results showed that libido score and physiological changes were not affected by treatments and time periods. Anejaculation was observed in 11 (91.7%), 5 (41.7%) and 1 (8.3%) males at 3, 6 and 24 h post-tamsulosin injection respectively. The incidence returned to normal when compared with control groups at 24 h. The percentages of motile and live spermatozoa at 6 h post-tamsulosin injection were significantly lower (P < 0.05) than that of normal saline group. At 24 h post-injection, there were no significant differences of all semen parameters among treatments. This study demonstrated that tamsulosin had temporary effects on ejaculation and semen quality without reducing sex desire and physiological functions in male goats.

  11. Values of Alpha 1 Microglobulin Does Not Differ between Individuals with and without Family History of Balkan Endemic Nephropathy

    PubMed Central

    Trnacevic, Senaid; Hodzic, Emir; Atic, Mirza; Dugonjic, Maida; Hasanovic, Evlijana

    2014-01-01

    Aim. The aim of this study was to compare urinary alpha 1 microglobulin (A1MG) in healthy individuals with and without family burden for Balkan endemic nephropathy (BEN) in an endemic village. Methods. Otherwise healthy inhabitants with microalbuminuria or proteinuria were divided into two groups: with (n = 24) and without (n = 32) family BEN burden and screened for urinary A1MG and A1MG/urine creatinine ratio. Results. Average value of urinary A1MG was 10.35 ± 7.01 mg/L in group with and 10.79 ± 8.27 mg/L in group without family history for BEN (NS, P = 0.87). A1MG was higher than 10 mg/L in eight (33.33%) inhabitants with family history and in 12 (37.5%) without (NS, P = 0.187). Average values of urinary A1MG/creatinine ratio were 1.30 ± 1.59 and 0.94 ± 0.78 in group with and group without family BEN history (NS, P = 0.39, resp.). Elevated values of this ratio were found in 13 (54.17%) inhabitants with and 14 (43.75%) without family history for BEN (NS, P = 0.415). Conclusion. We did not find statistically significant difference in the examined markers between healthy individuals with and without family burden for BEN. We concluded that these markers are not predictive of risk for BEN. PMID:24563783

  12. Determination of human serum alpha1-acid glycoprotein and albumin binding of various marketed and preclinical kinase inhibitors.

    PubMed

    Zsila, Ferenc; Fitos, Ilona; Bencze, Gyula; Kéri, György; Orfi, László

    2009-01-01

    There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.

  13. Reaction of germinal centers in the T-cell-independent response to the bacterial polysaccharide alpha(1-->6)dextran.

    PubMed Central

    Wang, D; Wells, S M; Stall, A M; Kabat, E A

    1994-01-01

    Primary immunization of BALB/c mice with alpha(1-->6)dextran (DEX), a native bacterial polysaccharide, induces an unexpected pattern of splenic B-cell responses. After a peak of antibody-secreting B-cell response at day 4, deposition of dextran-anti-dextran immune complexes, as revealed by staining with both dextran and antibodies to dextran, occurs and persists in splenic follicles until at least the fourth week after immunization. Antigen-specific B cells appear and proliferate in such follicles, leading by day 11 to development of DEX-specific germinal centers as characterized by the presence of distinct regions of DEX+ peanut agglutinin-positive (PNA+) cells. At this time, fluorescence-activated cell sorter analysis also reveals the appearance of a distinct population of DEX+ PNA+ splenic B cells. In contrast, DEX+ PNA- cells, characterized by intense cytoplasmic staining, are present outside of splenic follicles, peak at day 4 to day 5, and persist until at least day 28. The frequency of these cells correlates with DEX-specific antibody-secreting cells, as detected by the ELISA-spot assay. Thus, in addition to the expected plasma cellular response, the typical T-cell-independent type II antigen, DEX, surprisingly also elicits the formation of antigen-specific germinal centers. These observations raise fundamental questions about the roles of germinal centers in T-cell-independent immune responses. Images PMID:7511812

  14. Collagen, type XI, alpha 1: an accurate marker for differential diagnosis of breast carcinoma invasiveness in core needle biopsies.

    PubMed

    Freire, Javier; Domínguez-Hormaetxe, Saioa; Pereda, Saray; De Juan, Ana; Vega, Alfonso; Simón, Laureano; Gómez-Román, Javier

    2014-12-01

    Accurate diagnosis of invasive breast lesions, when analyzed by Core Needle Biopsy, may suppose a major challenge for the pathologist. Various markers of invasiveness such as laminin, S-100 protein, P63 or calponin have been described; however, none of them is completely reliable. The use of a specific marker of the infiltrating tumor microenvironment seems vital to support the diagnosis of invasive against in situ lesions. At this point, Collagen, type XI, alpha 1 (COL11A1), might be helpful since it has been described to be associated to cancer associated fibroblasts in other tumors such as lung, pancreas or colorectal. This paper aims to analyze the role of COL11A1 as a marker of invasiveness in breast tumor lesions. Two hundred and one breast Core Needle Biopsy samples were analyzed by immunohistochemistry against pro-COL11A1. The results show a significant difference (p < 0.0001) when comparing the expression in infiltrative tumors (93%) versus immunostaining of non-invasive lesions (4%). Forty cases of underestimated DCIS were also stained for COL11A1, presenting a sensitivity of 90% when compared with p63 and calponin which not tagged invasion. In conclusion, pro-COL11A1 expression is a promising marker of invasive breast lesions, and may be included in immunohistochemical panels aiming at identifying infiltration in problematic breast lesions.

  15. Association between alpha-1 antichymotrypsin gene A/T polymorphism and primary intracerebral hemorrhage: a meta-analysis.

    PubMed

    Ye, Zusen; Ye, Qiang; Shao, Bei; He, Jincai; Zhu, Zhenguo; Cheng, Jianhua; Chen, Yanyan; Chen, Siyan; Huang, Xiaoya

    2015-01-01

    The present study is to use meta-analysis to explain the association between alpha-1 antichymotrypsin (ACT) gene A/T polymorphism and the risk of primary intracerebral hemorrhage (PICH). Relevant studies before 1 June 2015 were identified by searching PubMed, Cochrane database and Science Citation Index Expanded (SCIE), and the references of retrieved articles. Pooled odds ratios (ORs) with corresponding 95% confidence intervals (95% CIs) were used to assess the strength of the association. Five independent publications, with 774 PICH cases and 940 controls, were included. There was no statistical evidence of association between ACT polymorphism and PICH risk under all genetic models in overall estimates (allele model: OR = 1.01, 95% CI = 0.80-1.28; heterozygote model: OR = 0.93, 95% CI = 0.60-1.45; homozygote model: OR = 1.03, 95% CI = 0.59-1.80; dominant model: OR = 0.97, 95% CI = 0.65-1.46; recessive model: OR = 1.06, 95% CI = 0.72-1.57). No association was found in subgroup analysis based on ethnicity, Hardy-Weinberg equilibrium, location of hematoma and blood pressure. Sensitivity analysis suggested that the combined results were stable and reliable. No significant publication bias was found by Begg's test and Egger's regression test. The results of our meta-analysis indicate that ACT polymorphism is unlikely to contribute to PICH susceptibility. PMID:26885003

  16. Association between alpha-1 antichymotrypsin gene A/T polymorphism and primary intracerebral hemorrhage: a meta-analysis

    PubMed Central

    Ye, Zusen; Ye, Qiang; Shao, Bei; He, Jincai; Zhu, Zhenguo; Cheng, Jianhua; Chen, Yanyan; Chen, Siyan; Huang, Xiaoya

    2015-01-01

    The present study is to use meta-analysis to explain the association between alpha-1 antichymotrypsin (ACT) gene A/T polymorphism and the risk of primary intracerebral hemorrhage (PICH). Relevant studies before 1 June 2015 were identified by searching PubMed, Cochrane database and Science Citation Index Expanded (SCIE), and the references of retrieved articles. Pooled odds ratios (ORs) with corresponding 95% confidence intervals (95% CIs) were used to assess the strength of the association. Five independent publications, with 774 PICH cases and 940 controls, were included. There was no statistical evidence of association between ACT polymorphism and PICH risk under all genetic models in overall estimates (allele model: OR = 1.01, 95% CI = 0.80-1.28; heterozygote model: OR = 0.93, 95% CI = 0.60-1.45; homozygote model: OR = 1.03, 95% CI = 0.59-1.80; dominant model: OR = 0.97, 95% CI = 0.65-1.46; recessive model: OR = 1.06, 95% CI = 0.72-1.57). No association was found in subgroup analysis based on ethnicity, Hardy-Weinberg equilibrium, location of hematoma and blood pressure. Sensitivity analysis suggested that the combined results were stable and reliable. No significant publication bias was found by Begg’s test and Egger’s regression test. The results of our meta-analysis indicate that ACT polymorphism is unlikely to contribute to PICH susceptibility. PMID:26885003

  17. Yeast Mutants Deficient in Er-Associated Degradation of the Z Variant of Alpha-1-Protease Inhibitor

    PubMed Central

    McCracken, A. A.; Karpichey, I. V.; Ernaga, J. E.; Werner, E. D.; Dillin, A. G.; Courchesne, W. E.

    1996-01-01

    Saccharomyces cerevisiae mutants deficient in degradation of alpha-1-proteinase inhibitor Z (A1PiZ) have been isolated and genetically characterized. Wild-type yeast expressing A1PiZ synthesize an ER form of this protein that is rapidly degraded by an intracellular proteolytic process known as ER-associated protein degradation (ERAD). The mutant strains were identified after treatment with EMS using a colony blot immunoassay to detect colonies that accumulated high levels of A1PiZ. A total of 120,000 colonies were screened and 30 putative mutants were identified. The level of A1PiZ accumulation in these mutants, measured by ELISA, ranged from two to 11 times that of A1PiZ in the parent strain. Further studies demonstrated that the increased levels of A1PiZ in most of the mutant strains was not the result of defective secretion or elevated A1PiZ mRNA. Pulse chase experiments indicated that A1PiZ was stabilized in several strains, evidence that these mutants are defective in ER-associated protein degradation. Genetic analyses revealed that most of the mutations were recessive, ~30% of the mutants characterized conformed to simple Mendelian inheritance, and at least seven complementation groups were identified. PMID:8978025

  18. Co-expression of alpha(1,3)galactosyltransferase and Bacillus thuringiensis PIPLC enhances hyperacute rejection of tumor cells.

    PubMed

    Hellrung, Daniel J; Kisselev, Serguei; Link, Charles J

    2007-01-01

    The use of alpha(1,3)galactosyltransferase (alphaGT) as a method of inducing hyperacute rejection of tumors has been gaining interest recently. However, the approach is based in part on the sensitivity of each tumor line to the effects of complement lysis. Tumors expressing complement resistance factors such as membrane cofactor (CD46), decay accelerating factor (CD55) and protectin (CD59) have been shown to be more resistant to complement mediated lysis. Anchored to the membrane by a glycosylphosphoinositol moiety (GPI-anchored), CD55 and CD59 can be cleaved by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PIPLC). Complement resistant A549 human lung carcinoma cells were engineered to express both the murine alphaGT gene and the B. thuringiensis PIPLC gene to alleviate complement resistance and enhance alphagal-mediated cancer killing. The PIPLC native signal sequence was replaced with the human epidermal growth factor signal sequence, EGFssPIPLC, to induce secretion from A549. Expression of EGFssPIPLC resulted in complete removal of CD55 and CD59 while sparing the non-GPI-anchored CD46. Results demonstrated that A549 cells transduced with two recombinant retroviral vectors carrying the alphaGT and EGFssPIPLC genes expressed high levels of alphagal epitope and exhibited a 5-fold increase in sensitivity to anti-alphagal mediated complement lysis.

  19. Accessibilome of human glioblastoma: collagen-VI-alpha-1 is a new target and a marker of poor outcome.

    PubMed

    Turtoi, Andrei; Blomme, Arnaud; Bianchi, Elettra; Maris, Pamela; Vannozzi, Riccardo; Naccarato, Antonio Giuseppe; Delvenne, Philippe; De Pauw, Edwin; Bevilacqua, Generoso; Castronovo, Vincent

    2014-12-01

    Functional targeted therapy has unfortunately failed to improve the outcome of glioblastoma patients. Success stories evidenced by the use of antibody-drug conjugates in other tumor types are encouraging, but targets specific to glioblastoma and accessible through the bloodstream remain scarce. In the current work, we have identified and characterized novel and accessible proteins using an innovative proteomic approach on six human glioblastomas; the corresponding data have been deposited in the PRIDE database identifier PXD001398. Among several clusters of uniquely expressed proteins, we highlight collagen-VI-alpha-1 (COL6A1) as a highly expressed tumor biomarker with low levels in most normal tissues. Immunohistochemical analysis of glioma samples from 61 patients demonstrated that COL6A1 is a significant and consistent feature of high-grade glioma. Deposits of COL6A1 were evidenced in the perivascular regions of the tumor-associated vasculature and in glioma cells found in pseudopalisade structures. Retrospective analysis of public gene-expression data sets from over 300 glioma patients demonstrated a significant correlation of poor patient outcome and high COL6A1 expression. In a proof-of-concept study, we use chicken chorioallantoic membrane in vivo model to show that COL6A1 is a reachable target for IV-injected antibodies. The present data warrant further development of human COL6A1 antibodies for assessing the quantitative biodistribution in the preclinical tumor models. PMID:25325876

  20. The effect of alpha 1-blocker, bunazosin on a murine model of congestive heart failure induced by viral myocarditis.

    PubMed

    Yamada, T; Matsumori, A; Okada, I; Tominaga, M; Kawai, C

    1992-11-01

    The purpose of this study was to investigate the therapeutic effect of an alpha 1-blocker, bunazosin, using an experimental murine model of congestive heart failure induced by viral myocarditis. This model is characterized by a high incidence of severe myocarditis and subsequent congestive heart failure, and is suitable for the evaluation of the effect of drugs. To estimate myocardial damage objectively and quantitatively, we used antimyosin monoclonal antibody in addition to histopathological grading. Four-week-old BALB/c mice were inoculated with encephalomyocarditis virus. The mice were injected daily with bunazosin or saline as a placebo from the day of viral inoculation until day 7 (protocol-I) or day 14 (protocol-II), or from day 4 to day 14 (protocol-III). They were then injected with 1.5 microCi of indium-111 labeled antimyosin antibody and were killed 24 h later. The antimyosin cardiac uptake was counted and histopathological grading was performed. The heart-weight to body-weight ratio, left ventricular dimension, histopathological grades and antimyosin cardiac uptake were significantly lower in the bunazosin group than in the placebo group in protocol-II, but not in protocol-I or protocol-III. Bunazosin showed a protective effect against viral myocarditis only when it was started early after infection and continued until the stage of congestive heart failure.

  1. Pulmonary artery rupture during Swan-Ganz catheterisation: a case report.

    PubMed

    Villaverde, Raquel Vilariño; Vanhaebost, Jessica; Grabherr, Silke; Palmiere, Cristian

    2014-03-01

    Catheter-induced pulmonary artery rupture is an infrequent complication that may occur during invasive cardiopulmonary monitoring. Fatal cases are uncommon and result from hemoptysis and flooding of the opposite lung with resulting hypoyxia. Alpha-1-antitrypsin deficiency is a rare genetic disorder characterised by low serum levels of alpha-1-antitrypsin, critical in maintaining connective tissue integrity. Besides pulmonary emphysema, recent observations suggest that alpha-1-antitrypsin deficiency may also be involved in vascular wall weakening, thereby predisposing arteries to dissection and aneurysm formation. In this article, we describe an autopsy case of pulmonary artery iatrogenic rupture due to insertion of a Swan-Ganz catheter in an 82-year-old woman suffering from pulmonary hypertension and alpha-1-antitrypsin deficiency. The exact source of bleeding could not be precisely identified during autopsy due to the extent of tissue hemorrhage, though postmortem angiography revealed a contrast medium extravasation from a branch of the left pulmonary lower lobar artery. The case herein emphasises the importance of postmortem angiography in facilitating the detection of vascular injuries, the importance of familiarity with intensive care techniques and procedures on behalf of forensic pathologists as well as in-depth knowledge of all possible contributing conditions and predisposing disorders in the pathogenesis of death.

  2. Deficient and Null Variants of SERPINA1 Are Proteotoxic in a Caenorhabditis elegans Model of α1-Antitrypsin Deficiency.

    PubMed

    Cummings, Erin E; O'Reilly, Linda P; King, Dale E; Silverman, Richard M; Miedel, Mark T; Luke, Cliff J; Perlmutter, David H; Silverman, Gary A; Pak, Stephen C

    2015-01-01

    α1-antitrypsin deficiency (ATD) predisposes patients to both loss-of-function (emphysema) and gain-of-function (liver cirrhosis) phenotypes depending on the type of mutation. Although the Z mutation (ATZ) is the most prevalent cause of ATD, >120 mutant alleles have been identified. In general, these mutations are classified as deficient (<20% normal plasma levels) or null (<1% normal levels) alleles. The deficient alleles, like ATZ, misfold in the ER where they accumulate as toxic monomers, oligomers and aggregates. Thus, deficient alleles may predispose to both gain- and loss-of-function phenotypes. Null variants, if translated, typically yield truncated proteins that are efficiently degraded after being transiently retained in the ER. Clinically, null alleles are only associated with the loss-of-function phenotype. We recently developed a C. elegans model of ATD in order to further elucidate the mechanisms of proteotoxicity (gain-of-function phenotype) induced by the aggregation-prone deficient allele, ATZ. The goal of this study was to use this C. elegans model to determine whether different types of deficient and null alleles, which differentially affect polymerization and secretion rates, correlated to any extent with proteotoxicity. Animals expressing the deficient alleles, Mmalton, Siiyama and S (ATS), showed overall toxicity comparable to that observed in patients. Interestingly, Siiyama expressing animals had smaller intracellular inclusions than ATZ yet appeared to have a greater negative effect on animal fitness. Surprisingly, the null mutants, although efficiently degraded, showed a relatively mild gain-of-function proteotoxic phenotype. However, since null variant proteins are degraded differently and do not appear to accumulate, their mechanism of proteotoxicity is likely to be different to that of polymerizing, deficient mutants. Taken together, these studies showed that C. elegans is an inexpensive tool to assess the proteotoxicity of different AT

  3. Possible Role of α1-Antitrypsin in Endometriosis-Like Grafts From a Mouse Model of Endometriosis.

    PubMed

    Tamura, Kazuhiro; Takashima, Haruka; Fumoto, Keiko; Kajihara, Takeshi; Uchino, Satomi; Ishihara, Osamu; Yoshie, Mikihiro; Kusama, Kazuya; Tachikawa, Eiichi

    2015-09-01

    Previous study indicated that bleeding into the peritoneum may accelerate inflammatory response in endometriosis-like grafts in mice. To identify changes in protein levels in the grafts from mice that underwent unilateral ovariectomy (uOVX), which causes bleeding from ovarian arteries and vein, the grafts were generated by injecting a suspension of human endometrial cells in BALB/c nude female mice, and protein profile changes were compared with non-uOVX control mice. The level of α1-antitrypsin (α1-AT) decreased in grafts from nude mice that underwent uOVX. The levels of phosphorylated Akt, mammalian target of rapamycin, S6K, regulatory factors for cell survival, and of phosphorylated nuclear factor κB, an inflammatory mediator, were higher in endometriosis-like grafts from the uOVX group than from the control. The grafts were mostly comprised of stromal cells. The bioactivity of α1-AT was assessed by investigating cytokine expression in protease-activated receptor (PAR) 1/2 agonists-stimulated stromal cells. The PARs promoted the expression of interleukin 8 (IL-8), but treatment with α1-AT blocked IL-8 expression dose dependently. Knocking down α1-AT expression increased the constitutive IL-6, IL-8, and cyclooxygenase 2 expression as well as PAR1 agonist-stimulated IL-6 expression. These findings support the notion that decreased α1-AT protein in the grafts constituted with human endometrial cells in mice may have exacerbated inflammation in endometriosis-like grafts, suggesting the possible involvement of α1-AT in the pathophysiology of endometriosis.

  4. Correlation Between Arteriosclerosis and Periodontal Condition Assessed by Lactoferrin and α1-Antitrypsin Levels in Gingival Crevicular Fluid.

    PubMed

    Hayashi, Shuji; Yamada, Hirotsugu; Fukui, Makoto; Ito, Hiro-o; Sata, Masataka

    2015-01-01

    Patients with periodontal disease exhibit exacerbated atherosclerosis, aortic stiffness, or vascular endothelial dysfunction. However, in a recent scientific statement, the American Heart Association noted that neither has periodontal disease been proven to cause atherosclerotic vascular disease nor has the treatment of periodontal disease been proven to prevent atherosclerotic vascular disease. Therefore, the aim of the present study was to examine the correlation between periodontal condition and arteriosclerosis in patients with coronary artery disease (CAD), which is usually accompanied by systemic arteriosclerosis.We measured levels of gingival crevicular fluid lactoferrin (GCF-Lf) and α1-antitrypsin (GCF-AT) in 72 patients (67 ± 8 years, 56 men) with CAD. Furthermore, we evaluated the maximum intima-media thickness (max IMT) and plaque score of the carotid arteries as well as brachial-ankle pulse wave velocity (baPWV) and flow-mediated dilation (FMD) of the brachial artery, each of which is a parameter for determining arteriosclerosis status. The average level of GCF-Lf was 0.29 ± 0.36 µg/mL and that of GCF-AT was 0.31 ± 0.66 µg/mL, with significant correlation between the two (r = 0.701, P < 0.001). No significant difference in GCF-Lf and GCF-AT levels was observed between patients with single-, double-, and triple-vessel CAD. There were no significant correlations between the arteriosclerosis parameters (ie, max IMT, plaque score, baPWV, and FMD) and GCF-Lf or GCF-AT.No correlation between the GCF biomarkers and the severity of arteriosclerosis was detected. This result may suggest that worsening of the periodontal condition assessed by GCF biomarkers is not a major potential risk factor for arteriosclerosis.

  5. Pre- and neonatal exposure of the Dahl rat to NaCl: development and regional distribution of myocardial alpha 1-adrenergic and cholinergic receptor sites.

    PubMed

    McCaughran, J A; Juno, C J; Friedman, R

    1986-06-01

    The prenatal and/or postweaning effects of a hypertensinogenic high NaCl-containing diet (8.0% NaCl, w/w) on (1) the regional distribution of alpha 1-adrenoceptors and muscarinic cholinergic receptor sites in the heart and (2) the predisposition/resistance to hypertension (HT) were assessed in the inbred Dahl HT-sensitive (S/JR) and HT-resistant (R/JR) rat. The density of alpha 1-adrenoceptors was reduced in the left ventricle but not consistently affected in the ventricular septum, right ventricle, or atria of S/JR offspring with NaCl-induced HT. Both normotensive and hypertensive S/JR rats also displayed a significantly greater density of cholinergic receptor sites in the atria but few consistent alterations in other regions of the heart, compared to R/JR rats. Maternal diet had no effect on the predisposition/resistance to salt-induced HT and little effect on the regional development of alpha 1-adrenoceptors and cholinergic receptor sites. The results of this study suggest that the reduced density of ventricular alpha 1-adrenoceptors in the S/JR strain is a consequence of HT while the elevated density of cholinergic receptors in the atria may be related to the genetic predisposition/resistance to HT.

  6. The human alpha 2(IV) collagen gene, COL4A2, is syntenic with the alpha 1(IV) gene, COL4A1, on chromosome 13.

    PubMed

    Solomon, E; Hall, V; Kurkinen, M

    1987-05-01

    We have previously assigned the gene for the alpha 1 chain of type IV collagen to chromosome 13. In this report we show that the gene coding for the second chain of this heterotrimer is on the same chromosome. This is the first example of the genes for both chains of one collagen molecule being syntenic. PMID:3674752

  7. Effects of alpha1-adrenergic receptor blockade by doxazosin on renin-angiotensin system-regulating aminopeptidase and vasopressin-degrading activities in male and female rat thalamus.

    PubMed

    de la Chica-Rodríguez, S; Cortés-Denia, P; Ramírez-Expósito, M J; Martínez-Martos, J M

    2007-11-01

    The thalamus has connections with central autonomic centers involved in cardiovascular control and is enervated by noradrenergic fibers. The excitability of thalamic neurons is due to a reduction of ionic currents mediated by alpha(1)-adrenoceptors. The brain renin- angiotensin system (RAS) and the peptide hormone arginine-vasopressin (AVP) are also involved in the central control of blood pressure, and fluid and electrolyte homeostasis. It has been extensively reported that aminopeptidase A (APA), aminopeptidase B (APB), aminopeptidase N (APN), and vasopressin-degrading cystyl aminopeptidase activity (AVP-DA) play an important role in the regulation of the activity of angiotensins and AVP. We have analyzed the effect of alpha(1)-adrenoceptor blockade by doxazosin on RAS-regulating aminopeptidase activities and AVP-DA in soluble and membrane-bound fractions of male and female rat thalamus. Our results show that alpha(1)-adrenoceptors blockade by doxazosin does not modify the RAS through its degrading peptidases at thalamic level either in male or female rats. However, alpha(1)-adrenoceptors blockade shows gender differences in AVP-DA, increasing in males but not in females, supporting an increased capacity of males against females to degrade AVP and, therefore, to regulate cardiovascular homeostasis, under this pharmacological manipulation.

  8. Rescue of type I collagen-deficient phenotype by retroviral-vector-mediated transfer of human pro alpha 1(I) collagen gene into Mov-13 cells.

    PubMed Central

    Stacey, A; Mulligan, R; Jaenisch, R

    1987-01-01

    A full-length cDNA clone corresponding to the human pro alpha 1(I) collagen gene was isolated and inserted into a retrovirus vector. Cell lines were obtained which produced recombinant viruses transducing the collagen cDNA (HUC virus). To test whether the transduced cDNA was functional, Mov-13 mouse cells were infected with the virus. These cells do not produce any type I collagen due to an insertional mutation of the pro alpha 1(I) gene which blocks transcription. While normal amounts of pro alpha 2(I) RNA were synthesized, no alpha 2(I) collagen chains were detectable in the mutant Mov-13 cells. Infection with HUC virus, however, resulted in the production of stable type I collagen, which was secreted into the medium. Analysis of pepsin-resistant proteins indicated that interspecies heterotrimers consisting of human alpha 1(I) and mouse alpha 2(I) collagen chains were secreted by the infected Mov-13 cells. Our results show that pro alpha (I) collagen chains from species as distant as human and mouse can associate to form stable type I collagen. The availability of a retrovirus vector transducing a functional pro alpha 1(I) collagen gene combined with the Mov-13 mutant system should enable us to study the effect of specific mutations on the synthesis, assembly, and function of type I collagen, not only in tissue culture but also in the animal. Images PMID:3599181

  9. Human collagen genes encoding basement membrane. cap alpha. 1(IV) and. cap alpha. 2(IV) chains map to the distal long arm of chromosome 13

    SciTech Connect

    Griffin, C.A.; Emanuel, B.S.; Hansen, J.R.; Cavenee, W.K.; Myers, J.C.

    1987-01-01

    At least 20 genes encode the structurally related collagen chains that comprise > 10 homo- or heterotrimeric types. Six members of this multigene family have been assigned to five chromosomes in the human genome. The two type I genes, ..cap alpha..1 and ..cap alpha..2, are located on chromosomes 17 and 7, respectively, and the ..cap alpha..1(II) gene is located on chromosome 12. Their recent mapping of the ..cap alpha..1(III) and ..cap alpha..2(V) genes to the q24.3 ..-->.. q31 region of chromosome 2 provided the only evidence that the collagen genes are not entirely dispersed. To further determine their organization, the authors and others localized the ..cap alpha..1(IV) gene to chromosome 13 and in their experiments sublocalized the gene to band q34 by in situ hybridization. Here they show the presence of the ..cap alpha..2 type IV locus also on the distal long arm of chromosome 13 by hybridizing a human ..cap alpha..2(IV) cDNA clone to rodent-human hybrids and to metaphase chromosomes. These studies represent the only demonstration of linkage between genes encoding both polypeptide chains of the same collagen type.

  10. Molecular cloning and characterization of a plant alpha1,3/4-fucosidase based on sequence tags from almond fucosidase I.

    PubMed

    Zeleny, Reinhard; Leonard, Renaud; Dorfner, Georg; Dalik, Thomas; Kolarich, Daniel; Altmann, Friedrich

    2006-04-01

    Our work with almond peptide N-glycosidase A made us interested also in the alpha1,3/4-fucosidase which is used as a specific reagent for glycoconjugate analysis. The enzyme was purified to presumed homogeneity by a series of chromatographic steps including dye affinity and fast-performance anion exchange chromatography. The 63 kDa band was analyzed by tandem mass spectrometry which yielded several partial sequences. A homology search retrieved the hypothetical protein Q8GW72 from Arabidopsis thaliana. This protein has recently been described as being specific for alpha1,2-linkages. However, cDNA cloning and expression in Pichia pastoris of the A. thaliana fucosidase showed that it hydrolyzed fucose in 3- and 4-linkage to GlcNAc in Lewis determinants whereas neither 2-linked fucose nor fucose in 3-linkage to the innermost GlcNAc residue were attacked. This first cloning of a plant alpha1,3/4-fucosidase also confirmed the identity of the purified almond enzyme and thus settles the notorious uncertainty about its molecular mass. The alpha1,3/4-fucosidase from Arabidopsis exhibited striking sequence similarity with an enzyme of similar substrate specificity from Streptomyces sp. (Q9Z4I9) and with putative proteins from rice. PMID:16516937

  11. Homology modeling of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABA receptor channels and Surflex-docking of fipronil.

    PubMed

    Cheng, Jin; Ju, Xiu-Lian; Chen, Xiang-Yang; Liu, Gen-Yan

    2009-09-01

    To further explore the mechanism of selective binding of the representative gamma-aminobutyric acid receptors (GABARs) noncompetitive antagonist (NCA) fipronil to insect over mammalian GABARs, three-dimensional models of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABAR were generated by homology modeling, using the cryo-electron microscopy structure of the nicotinic acetylcholine receptor (nAChR) of Torpedo marmorata as a template. Fipronil was docked into the putative binding site of the human alpha 1 beta 2 gamma 2 and house fly beta 3 receptors by Surflex-docking, and the calculated docking energies are in agreement with experimental results. The GABA receptor antagonist fipronil exhibited higher potency with house fly beta 3 GABAR than with human alpha 1 beta 2 gamma 2 GABAR. Furthermore, analyses of Surflex-docking suggest that the H-bond interaction of fipronil with Ala2 and Thr6 in the second transmembrane segment (TM2) of these GABARs plays a relatively important role in ligand selective binding. The different subunit assemblies of human alpha 1 beta 2 gamma 2 and house fly beta 3 GABARs may result in differential selectivity for fipronil.

  12. The primary structure of water buffalo alpha(s1)- and beta-casein identification of phosphorylation sites and characterization of a novel beta-casein variant.

    PubMed

    Ferranti, P; Scaloni, A; Caira, S; Chianese, L; Malorni, A; Addeo, F

    1998-11-01

    The primary structure of water buffalo alpha(s1)-casein and of beta-casein A and B variants has been determined using a combination of mass spectrometry and Edman degradation procedures. The phosphorylated residues were localized on the tryptic phosphopeptides after performing a beta-elimination/thiol derivatization. Water buffalo alpha(s1)-casein, resolved in three discrete bands by isoelectric focusing, was found to consist of a single protein containing eight, seven, or six phosphate groups. Compared to bovine alpha(s1)-casein C variant, the water buffalo alpha(s1)-casein presented ten amino acid substitutions, seven of which involved charged amino acid residues. With respect to bovine betaA2-casein variant, the two water buffalo beta-casein variants A and B presented four and five amino acid substitutions, respectively. In addition to the phosphoserines, a phosphothreonine residue was identified in variant A. From the phylogenetic point of view, both water buffalo beta-casein variants seem to be homologous to bovine betaA2-casein.

  13. Modulation of transcriptional activation and coactivator interaction by a splicing variation in the F domain of nuclear receptor hepatocyte nuclear factor 4alpha1.

    PubMed

    Sladek, F M; Ruse, M D; Nepomuceno, L; Huang, S M; Stallcup, M R

    1999-10-01

    Transcription factors, such as nuclear receptors, often exist in various forms that are generated by highly conserved splicing events. Whereas the functional significance of these splicing variants is often not known, it is known that nuclear receptors activate transcription through interaction with coactivators. The parameters, other than ligands, that might modulate those interactions, however, are not well characterized, nor is the role of splicing variants. In this study, transient transfection, yeast two-hybrid, and GST pulldown assays are used to show not only that nuclear receptor hepatocyte nuclear factor 4 alpha1 (HNF4alpha1, NR2A1) interacts with GRIP1, and other coactivators, in the absence of ligand but also that the uncommonly large F domain in the C terminus of the receptor inhibits that interaction. In vitro, the F domain was found to obscure an AF-2-independent binding site for GRIP1 that did not map to nuclear receptor boxes II or III. The results also show that a natural splicing variant containing a 10-amino-acid insert in the middle of the F domain (HNF4alpha2) abrogates that inhibition in vivo and in vitro. A series of protease digestion assays indicates that there may be structural differences between HNF4alpha1 and HNF4alpha2 in the F domain as well as in the ligand binding domain (LBD). The data also suggest that there is a direct physical contact between the F domain and the LBD of HNF4alpha1 and -alpha2 and that that contact is different in the HNF4alpha1 and HNF4alpha2 isoforms. Finally, we propose a model in which the F domain of HNF4alpha1 acts as a negative regulatory region for transactivation and in which the alpha2 insert ameliorates the negative effect of the F domain. A conserved repressor sequence in the F domains of HNF4alpha1 and -alpha2 suggests that this model may be relevant to other nuclear receptors as well. PMID:10490591

  14. Time-dependent changes in the expression of thyroid hormone receptor alpha 1 in the myocardium after acute myocardial infarction: possible implications in cardiac remodelling.

    PubMed

    Pantos, Constantinos; Mourouzis, Iordanis; Xinaris, Christodoulos; Kokkinos, Alexandros D; Markakis, Konstantinos; Dimopoulos, Antonios; Panagiotou, Matthew; Saranteas, Theodosios; Kostopanagiotou, Georgia; Cokkinos, Dennis V

    2007-04-01

    The present study investigated whether changes in thyroid hormone (TH) signalling can occur after acute myocardial infarction (AMI) with possible physiological consequences on myocardial performance. TH may regulate several genes encoding important structural and regulatory proteins particularly through the TR alpha 1 receptor which is predominant in the myocardium. AMI was induced in rats by ligating the left coronary artery while sham-operated animals served as controls. This resulted in impaired cardiac function in AMI animals after 2 and 13 weeks accompanied by a shift in myosin isoforms expression towards a fetal phenotype in the non-infarcted area. Cardiac hypertrophy was evident in AMI hearts after 13 weeks but not at 2 weeks. This response was associated with a differential pattern of TH changes at 2 and 13 weeks; T(3) and T(4) levels in plasma were not changed at 2 weeks but T(3) was significantly lower and T(4) remained unchanged at 13 weeks. A twofold increase in TR alpha 1 expression was observed after 13 weeks in the non-infarcted area, P<0.05 versus sham operated, while TR alpha 1 expression remained unchanged at 2 weeks. A 2.2-fold decrease in TR beta 1 expression was found in the non-infarcted area at 13 weeks, P<0.05, while no change in TR beta 1 expression was seen at 2 weeks. Parallel studies with neonatal cardiomyocytes showed that phenylephrine (PE) administration resulted in 4.5-fold increase in the expression of TR alpha 1 and 1.6-fold decrease in TR beta 1 expression versus untreated, P<0.05. In conclusion, cardiac dysfunction which occurs at late stages after AMI is associated with increased expression of TR alpha 1 receptor and lower circulating tri-iodothyronine levels. Thus, apo-TR alpha 1 receptor state may prevail contributing to cardiac fetal phenotype. Furthermore, down-regulation of TR beta 1 also contributes to fetal phenotypic changes. alpha1-adrenergic signalling is, at least in part, involved in this response.

  15. An upstream regulatory region mediates high-level, tissue-specific expression of the human alpha 1(I) collagen gene in transgenic mice.

    PubMed Central

    Slack, J L; Liska, D J; Bornstein, P

    1991-01-01

    Studies in vitro have not adequately resolved the role of intronic and upstream elements in regulating expression of the alpha 1(I) collagen gene. To address this issue, we generated 12 separate lines of transgenic mice with alpha 1(I) collagen-human growth hormone (hGH) constructs containing different amounts of 5'-flanking sequence, with or without most of the first intron. Transgenes driven by 2.3 kb of alpha 1(I) 5'-flanking sequence, whether or not they contained the first intron, were expressed at a high level and in a tissue-specific manner in seven out of seven independent lines of transgenic mice. In most tissues, the transgene was expressed at levels approaching that of the endogenous alpha 1(I) gene and was regulated identically with the endogenous gene as animals aged. However, in lung, expression of the transgene was anomalously high, and in muscle, expression was lower than that of the endogenous gene, suggesting that in these tissues other regions of the gene may participate in directing appropriate expression. Five lines of mice were generated containing transgenes driven by 0.44 kb of alpha 1(I) 5'-flanking sequence (with or without the first intron), and expression was detected in four out of five of these lines. The level of expression of the 0.44-kb constructs in the major collagen-producing tissues was 15- to 500-fold lower than that observed with the longer 2.3-kb promoter. While transgenes containing the 0.44-kb promoter and the first intron retained a modest degree of tissue-specific expression, those without the first intron lacked tissue specificity and were poorly expressed in all tissues except lung.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2005897

  16. Imidazenil: a low efficacy agonist at alpha1- but high efficacy at alpha5-GABAA receptors fail to show anticonvulsant cross tolerance to diazepam or zolpidem.

    PubMed

    Auta, James; Impagnatiello, Francesco; Kadriu, Bashkim; Guidotti, Alessandro; Costa, Erminio

    2008-08-01

    Whereas advances in the molecular biology of GABA(A) receptor complex using knock-out and knock-in mice have been valuable in unveiling the structure, composition, receptor assembly, and several functions of different GABA(A) receptor subtypes, the mechanism(s) underlying benzodiazepine (BZ) tolerance and withdrawal remain poorly understood. Studies using specific GABA(A) receptor subunit knock-in mice suggest that tolerance to sedative action of diazepam requires long-term activation of alpha1 and alpha5 GABA(A) receptor subunits. We investigated the role of long-term activation of these GABA(A) receptor subunits during anticonvulsant tolerance using high affinity and high intrinsic efficacy ligands for GABA(A) receptors expressing the alpha5 subunit (imidazenil) or alpha1 subunit (zolpidem), and a non-selective BZ recognition site ligand (diazepam). We report here that long-term activation of GABA(A) receptors by zolpidem and diazepam but not by imidazenil elicits anticonvulsant tolerance. Although anticonvulsant cross-tolerance occurs between diazepam and zolpidem, there is no cross-tolerance between imidazenil and diazepam or zolpidem. Furthermore, diazepam or zolpidem long-term treatment decreased the expression of mRNA encoding the alpha1 GABA(A) receptor subunit in prefrontal cortex by 43% and 20% respectively. In addition, diazepam but not zolpidem long-term treatment produced a 30% increase in the expression of the alpha5 GABA(A) receptor subunit mRNA in prefrontal cortex. In contrast, imidazenil which is devoid of anticonvulsant tolerance does not elicit significant changes in the expression of alpha1 or alpha5 GABA(A) receptor subunit. These findings suggest that long-term activation of GABA(A) receptors containing the alpha1 or other subunits but not the alpha5 receptor subunit is essential for the induction of anticonvulsant tolerance.

  17. Mechanisms of postspaceflight orthostatic hypotension: low alpha1-adrenergic receptor responses before flight and central autonomic dysregulation postflight

    NASA Technical Reports Server (NTRS)

    Meck, Janice V.; Waters, Wendy W.; Ziegler, Michael G.; deBlock, Heidi F.; Mills, Paul J.; Robertson, David; Huang, Paul L.

    2004-01-01

    Although all astronauts experience symptoms of orthostatic intolerance after short-duration spaceflight, only approximately 20% actually experience presyncope during upright posture on landing day. The presyncopal group is characterized by low vascular resistance before and after flight and low norepinephrine release during orthostatic stress on landing day. Our purpose was to determine the mechanisms of the differences between presyncopal and nonpresyncopal groups. We studied 23 astronauts 10 days before launch, on landing day, and 3 days after landing. We measured pressor responses to phenylephrine injections; norepinephrine release with tyramine injections; plasma volumes; resting plasma levels of chromogranin A (a marker of sympathetic nerve terminal release), endothelin, dihydroxyphenylglycol (DHPG, an intracellular metabolite of norepinephrine); and lymphocyte beta(2)-adrenergic receptors. We then measured hemodynamic and neurohumoral responses to upright tilt. Astronauts were separated into two groups according to their ability to complete 10 min of upright tilt on landing day. Compared with astronauts who were not presyncopal on landing day, presyncopal astronauts had 1). significantly smaller pressor responses to phenylephrine both before and after flight; 2). significantly smaller baseline norepinephrine, but significantly greater DHPG levels, on landing day; 3). significantly greater norepinephrine release with tyramine on landing day; and 4). significantly smaller norepinephrine release, but significantly greater epinephrine and arginine vasopressin release, with upright tilt on landing day. These data suggest that the etiology of orthostatic hypotension and presyncope after spaceflight includes low alpha(1)-adrenergic receptor responsiveness before flight and a remodeling of the central nervous system during spaceflight such that sympathetic responses to baroreceptor input become impaired.

  18. Ranolazine enhances nicardipine-induced relaxation of alpha1-adrenoceptor-mediated contraction on isolated rabbit aorta.

    PubMed

    Malavaki, Christina; Hatziefthimiou, Apostolia; Daskalopoulou, Stella S; Stefanidis, Ioannis; Karatzaferi, Christina; Aidonidis, Isaac

    2015-04-01

    Ranolazine (RAN) and nicardipine (NIC) have been studied for their vasorelaxing effects but the combination of these agents against adrenergic vasoconstriction has not been tested. The present study aimed at investigating the vasorelaxing effect by the combination of the two agents on alpha1-adrenoceptor-mediated contraction on isolated rabbit aorta. Aortic rings were mounted for isometric tension recording in organ baths containing Krebs-Henseleit solution. Concentration-response curves of RAN (10(-9) to 10(-4) M), NIC (10(-1) to 10(-5) M), and RAN + NIC (3 x 10(-6) M) were obtained in a cumulative manner using phenylephrine (PE, 2 x 10(-6) M) as constrictor agent. The effective concentration (EC)50 values for RAN and NIC were 6.5 x 10(-6) M and 1.4 x 10(-5) M, respectively. The treatment of PE-precontracted aortic rings with either RAN or NIC up to 65 min revealed that both agents displayed a biphasic pattern of initial rising and late sustained phases of relaxation. At 35 min of incubation, RAN and NIC induced relaxation by 23 +/- 3% and 14 +/- 4%, respectively (N = 7, P=NS, RAN vs. NIC); their combination resulted in a 34 +/- 4% relaxation (N=7; P < 0.01, RAN + NIC vs. NIC). At 65 min the effect of NIC prevailed and tended to be closer to the values of the combination treatment (P < 0.01, RAN + NIC vs. RAN). The results indicate that RAN at therapeutic concentrations exerts a significant additive vasorelaxing effect when combined with NIC in rabbit aorta.

  19. Ranolazine enhances nicardipine-induced relaxation of alpha1-adrenoceptor-mediated contraction on isolated rabbit aorta.

    PubMed

    Malavaki, Christina; Hatziefthimiou, Apostolia; Daskalopoulou, Stella S; Stefanidis, Ioannis; Karatzaferi, Christina; Aidonidis, Isaac

    2015-04-01

    Ranolazine (RAN) and nicardipine (NIC) have been studied for their vasorelaxing effects but the combination of these agents against adrenergic vasoconstriction has not been tested. The present study aimed at investigating the vasorelaxing effect by the combination of the two agents on alpha1-adrenoceptor-mediated contraction on isolated rabbit aorta. Aortic rings were mounted for isometric tension recording in organ baths containing Krebs-Henseleit solution. Concentration-response curves of RAN (10(-9) to 10(-4) M), NIC (10(-1) to 10(-5) M), and RAN + NIC (3 x 10(-6) M) were obtained in a cumulative manner using phenylephrine (PE, 2 x 10(-6) M) as constrictor agent. The effective concentration (EC)50 values for RAN and NIC were 6.5 x 10(-6) M and 1.4 x 10(-5) M, respectively. The treatment of PE-precontracted aortic rings with either RAN or NIC up to 65 min revealed that both agents displayed a biphasic pattern of initial rising and late sustained phases of relaxation. At 35 min of incubation, RAN and NIC induced relaxation by 23 +/- 3% and 14 +/- 4%, respectively (N = 7, P=NS, RAN vs. NIC); their combination resulted in a 34 +/- 4% relaxation (N=7; P < 0.01, RAN + NIC vs. NIC). At 65 min the effect of NIC prevailed and tended to be closer to the values of the combination treatment (P < 0.01, RAN + NIC vs. RAN). The results indicate that RAN at therapeutic concentrations exerts a significant additive vasorelaxing effect when combined with NIC in rabbit aorta. PMID:26148375

  20. Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies.

    PubMed

    Bi, Cong; Jackson, Abby; Vargas-Badilla, John; Li, Rong; Rada, Giana; Anguizola, Jeanethe; Pfaunmiller, Erika; Hage, David S

    2016-05-15

    A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37°C for carbamazepine with AGP was found to be 1.0 (±0.5)×10(5)M(-1), which agreed with a previously reported value of 1.0 (±0.1)×10(5)M(-1). Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein.

  1. Serological activity against galactosyl-alpha(1-3)galactose in sera from patients with several kinetoplastida infections.

    PubMed Central

    Avila, J L; Rojas, M; Towbin, H

    1988-01-01

    Using rabbit erythrocyte-derived neutral glycosphingolipids enriched for a defined ceramide pentasaccharide as antigens, we have detected elevated anti-galactosyl-alpha(1-3)galactose (anti-G alpha G) antibody values in patients with American cutaneous leishmaniasis (ACL), chronic Chagas' disease, and Trypanosoma rangeli infections compared with normal subjects or with patients suffering from any of 15 other infectious diseases. The specificity of the G alpha G antibodies was determined by inhibition enzyme-linked immunosorbent assays, which revealed that several alpha-galactosyl- but not beta-galactosyl-bearing sugars blocked absorption of G alpha G antibodies to the specific antigen used. G alpha G antibodies were mainly distributed between immunoglobulin classes G and M in three Kinetoplastida infections studied, with a lower increase in reactivity detected in immunoglobulin A. Absorption of highly reactive G alpha G antibodies with purified murine laminin and nidogen, two basement membrane proteins, almost abolished G alpha G reactivity, suggesting the identity of anti-G alpha G with laminin and nidogen antibodies previously reported as elevated in Kinetoplastida infections. In ACL, G alpha G antibodies were detected in 71% of patients having skin lesions with a clinical evolution time of 0.5 month. This percentage increased with the time of evolution of skin lesions, reaching 93% in lesions older than 3 months, and tended to decrease inversely to the induration diameter in the skin leishmanin test. It is proposed that similar epitopes may exist on kinetoplast protozoa and that the determination of G alpha G antibodies may be a highly sensitive assay for the detection of humoral responses to Kinetoplastida infections. PMID:2449451

  2. An integrative approach combining ion mobility mass spectrometry, X-ray crystallography, and nuclear magnetic resonance spectroscopy to study the conformational dynamics of α1 -antitrypsin upon ligand binding.

    PubMed

    Nyon, Mun Peak; Prentice, Tanya; Day, Jemma; Kirkpatrick, John; Sivalingam, Ganesh N; Levy, Geraldine; Haq, Imran; Irving, James A; Lomas, David A; Christodoulou, John; Gooptu, Bibek; Thalassinos, Konstantinos

    2015-08-01

    Native mass spectrometry (MS) methods permit the study of multiple protein species within solution equilibria, whereas ion mobility (IM)-MS can report on conformational behavior of specific states. We used IM-MS to study a conformationally labile protein (α1 -antitrypsin) that undergoes pathological polymerization in the context of point mutations. The folded, native state of the Z-variant remains highly polymerogenic in physiological conditions despite only minor thermodynamic destabilization relative to the wild-type variant. Various data implicate kinetic instability (conformational lability within a native state ensemble) as the basis of Z α1 -antitrypsin polymerogenicity. We show the ability of IM-MS to track such disease-relevant conformational behavior in detail by studying the effects of peptide binding on α1 -antitrypsin conformation and dynamics. IM-MS is, therefore, an ideal platform for the screening of compounds that result in therapeutically beneficial kinetic stabilization of native α1 -antitrypsin. Our findings are confirmed with high-resolution X-ray crystallographic and nuclear magnetic resonance spectroscopic studies of the same event, which together dissect structural changes from dynamic effects caused by peptide binding at a residue-specific level. IM-MS methods, therefore, have great potential for further study of biologically relevant thermodynamic and kinetic instability of proteins and provide rapid and multidimensional characterization of ligand interactions of therapeutic interest.

  3. An integrative approach combining ion mobility mass spectrometry, X-ray crystallography, and nuclear magnetic resonance spectroscopy to study the conformational dynamics of α1-antitrypsin upon ligand binding

    PubMed Central

    Nyon, Mun Peak; Prentice, Tanya; Day, Jemma; Kirkpatrick, John; Sivalingam, Ganesh N; Levy, Geraldine; Haq, Imran; Irving, James A; Lomas, David A; Christodoulou, John; Gooptu, Bibek; Thalassinos, Konstantinos

    2015-01-01

    Native mass spectrometry (MS) methods permit the study of multiple protein species within solution equilibria, whereas ion mobility (IM)-MS can report on conformational behavior of specific states. We used IM-MS to study a conformationally labile protein (α1-antitrypsin) that undergoes pathological polymerization in the context of point mutations. The folded, native state of the Z-variant remains highly polymerogenic in physiological conditions despite only minor thermodynamic destabilization relative to the wild-type variant. Various data implicate kinetic instability (conformational lability within a native state ensemble) as the basis of Z α1-antitrypsin polymerogenicity. We show the ability of IM-MS to track such disease-relevant conformational behavior in detail by studying the effects of peptide binding on α1-antitrypsin conformation and dynamics. IM-MS is, therefore, an ideal platform for the screening of compounds that result in therapeutically beneficial kinetic stabilization of native α1-antitrypsin. Our findings are confirmed with high-resolution X-ray crystallographic and nuclear magnetic resonance spectroscopic studies of the same event, which together dissect structural changes from dynamic effects caused by peptide binding at a residue-specific level. IM-MS methods, therefore, have great potential for further study of biologically relevant thermodynamic and kinetic instability of proteins and provide rapid and multidimensional characterization of ligand interactions of therapeutic interest. PDB Code(s): 4PYW PMID:26011795

  4. Guinea-pig alpha 1-fetoprotein: purification, characterization, developmental and hormonal regulation, and behavior in diethylnitrosamine hepatocarcinogenesis.

    PubMed

    Gourdeau, H; Bélanger, L

    1983-10-01

    Physicochemical and immunological procedures were developed for the purification of guinea-pig alpha 1-fetoprotein (AFP). A double-antibody radioimmunoassay was developed which can detect 0.1 ng of AFP. The E1%278 nm of guinea-pig AFP is 6.6. Its molecular weight is 73 000. Its amino acid composition is similar to other AFP's, but it has a higher carbohydrate content (8.5%), owing to larger amounts of mannose and N-acetylglucosamine. Guinea-pig AFP has a slower electrophoretic mobility than other AFP's, owing to its more basic ionic structure. It separates into three electrophoretic variants in nondenaturing polyacrylamide gels; these are charge variants with isoelectric points of 5.0, 5.12, and 5.54. Changes in electrophoretic mobility after neuraminidase treatment indicate that charge heterogeneity is due to a variable content in sialic acid. Guinea-pig AFP has fatty-acid- and lectin-binding properties, but no affinity for sex hormones. Its biological half-life is 2.1 days. Its sites of synthesis are the liver and the yolk sac, with minor contributions by the upper gastrointestinal tract. AFP production appears to cease synchronously in the liver and yolk sac during the 8th week of gestation. From 4 to 8 weeks of gestation, AFP levels are at 2.5-3.5 mg/mL in fetal serum and 0.1-0.2 mg/mL in amniotic fluid. The proportion of electrophoretic and lectin-reactive AFP variants changes during development, reflecting the changing glycosylation of the protein. The gestational levels of AFP in the maternal compartment are indicative of a fetomaternal equilibration through transamniotic exchange. Serum AFP levels in normal adult guinea pigs are remarkably high, from 400 to 2000 ng/mL and occasionally up to over 5000 ng/mL. The administration of dexamethasone suppresses serum AFP levels. During liver carcinogenesis induced by diethylnitrosamine, serum AFP levels increase, moderately, when tumors develop.

  5. Effect of Silodosin, an Alpha1A-Adrenoceptor Antagonist, on Ventral Prostatic Hyperplasia in the Spontaneously Hypertensive Rat

    PubMed Central

    Shimizu, Shogo; Shimizu, Takahiro; Tsounapi, Panagiota; Higashi, Youichirou; Martin, Darryl T.; Nakamura, Kumiko; Honda, Masashi; Inoue, Keiji; Saito, Motoaki

    2015-01-01

    Background A decreased prostatic blood flow could be one of the risk factors for benign prostatic hyperplasia/benign prostatic enlargement. The spontaneously hypertensive rat (SHR) shows a chronic prostatic ischemia and hyperplastic morphological abnormalities in the ventral prostate. The effect of silodosin, a selective alpha1A-adrenoceptor antagonist, was investigated in the SHR prostate as a prostatic hyperplasia model focusing on prostatic blood flow. Methods Twelve-week-old male SHRs were administered perorally with silodosin (100 μg/kg/day) or vehicle once daily for 6 weeks. Wistar Kyoto (WKY) rats were used as normotensive controls and were treated with the vehicle. The effect of silodosin on blood pressure and prostatic blood flow were estimated and then the prostates were removed and weighed. The tissue levels of malondialdehyde (MDA), interleukin-6 (IL-6), chemokine (C-X-C motif) ligand 1/cytokine-induced neutrophil chemoattractant 1 (CXCL1/CINC1), tumor necrosis factor-alpha (TNF-α), transforming growth factor beta 1 (TGF-β1), basic fibroblast growth factor (bFGF) and alpha-smooth muscle actin (α-SMA) were measured. The histological evaluation was also performed by hematoxylin and eosin staining. Results There was a significant increase in blood pressure, prostate weight, prostate body weight ratio (PBR), tissue levels of MDA, IL-6, CXCL1/CINC1, TNF-α, TGF-β1, bFGF and α-SMA in the SHR compared to the WKY rat. The ventral prostate in the SHR showed the morphological abnormalities compared to the WKY rat. Prostatic blood flow was decreased in the SHR. However, treatment with silodosin significantly restored the decreased prostatic blood flow in the SHR. Moreover, silodosin normalized tissue levels of MDA, IL-6, CXCL1/CINC1, TNF-α, TGF-β1, bFGF and α-SMA, and it ameliorated ventral prostatic hyperplasia in the SHR excluding blood pressure. Silodosin decreased PBR but not prostate weight in the SHR. Conclusions Silodosin can inhibit the

  6. Epithelial but not stromal expression of collagen alpha-1(III) is a diagnostic and prognostic indicator of colorectal carcinoma.

    PubMed

    Wang, Xiao-Qing; Tang, Zu-Xiong; Yu, Dong; Cui, Shu-Jian; Jiang, Ying-Hua; Zhang, Qian; Wang, Jie; Yang, Peng-Yuan; Liu, Feng

    2016-02-23

    Colorectal cancer (CRC) is the third most common cancer in males and the second in females worldwide with very poor prognosis. Collagen alpha-1(III) (COL3A1) gene, encoding an extracellular matrix protein, is upregulated in human cancers. Here, we revealed that COL3A1 was increased in CRC by analysis of five Oncomine gene expression datasets (n = 496). Immunohistochemistry analysis of a tissue microarray (n = 90) demonstrated that cancer epithelial but not stromal COL3A1 was significantly upregulated comparing with the normal counterparts. High COL3A1 mRNA and/or protein expression was accompanied with high stage, T stage, Dukes stage, grade and older age, as well as smoking and recurrence status. Upregulated COL3A1 predicted poor overall (p = 0.003) and disease-free (p = 0.025) survival. Increased epithelial but not stromal COL3A1 protein predicted worse outcome (p = 0.03). Older patients (age>65) with high COL3A1 had worse survival than younger (age≤65) with high COL3A1. Plasma COL3A1 was increased in CRC patients (n = 86) by 5.4 fold comparing with healthy individuals, enteritis and polyps patients. Plasma COL3A1 had an area under curve (AUC) of 0.92 and the best sensitivity/specificity of 98.8%/69.1%. While plasma CEA had a poorer prediction power (AUC = 0.791, sensitivity/selectivity = 70.2%/73.0%). Older patients (age≥60) had higher plasma COL3A1 than younger patients. The epithelial COL3A1 protein had an AUC of 0.975 and the best sensitivity/specificity of 95.2%/91.1%. Silencing of COL3A1 suppressed CRC cell proliferation in in vitro MTT assay and in in vivo Zebra fish xenograft model by downregulation of PI3K/AKT and WNT signaling. COL3A1 was a novel diagnosis and prognosis marker of CRC. PMID:26741506

  7. Increased sensitization of acoustic startle response in spasmodic mice with a mutation of the glycine receptor alpha1-subunit gene.

    PubMed

    Plappert, C F; Pilz, P K; Becker, K; Becker, C M; Schnitzler, H U

    2001-06-01

    The spontaneous mutant mouse spasmodic (spd) carries a missense mutation affecting the glycine receptor alpha1-subunit gene. This results in a decreased binding affinity to glycine. Spd mutants show exaggerated acoustic startle responses (ASR). The present study sought to elucidate whether this increased ASR is due to a changed auditory processing or to stronger motor output resulting from a disinhibited motor system or, alternatively, to changes in modulatory influences on the startle pathway, namely in the mechanisms underlying habituation and sensitization. We found that in homozygous spd/spd mutants the startle threshold was lower, and the recorded slope of input/output (i/o) function, which reflects the relation between sensory input and motor output, was steeper. During repetitive presentation of high sound pressure level (SPL) startle stimuli (25 dB above startle threshold), ASR amplitudes did not decrease in spd/spd mutants as they do in the wildtype. In contrast, ASR amplitudes decreased when low SPL startle stimuli were presented. Footshocks presented after high SPL startle stimuli did not cause a further increase in ASR amplitudes of spd/spd mutants as in the wildtype. In heterozygous spd/+ mutants all these parameters were between those of spd/spd mutants and wildtype mice but closer to those of the wildtype. The steeper slope of i/o function in spd/spd mutants may be caused by both an increased sensory input and an increased motor output. The altered course of ASR amplitudes during repetitive stimulation and the deficit in additional footshock sensitization, however, can only be explained by an increased sensitization level in the spd/spd mutants. In accordance with the "dual process theory" strong sensitization evoked by high SPL startle stimuli supposedly counteracts habituation, leading to a constant high ASR amplitude. Furthermore, additional footshock sensitization is prevented. The increased sensitization level may be due to a change in auditory

  8. [beta]1-Adrenoceptor or [alpha]1-Adrenoceptor Activation Initiates Early Odor Preference Learning in Rat Pups: Support for the Mitral Cell/cAMP Model of Odor Preference Learning

    ERIC Educational Resources Information Center

    Harley, Carolyn W.; Darby-King, Andrea; McCann, Jennifer; McLean, John H.

    2006-01-01

    We proposed that mitral cell [beta]1-adrenoceptor activation mediates rat pup odor preference learning. Here we evaluate [beta]1-, [beta]2-, [alpha]1-, and [alpha]2-adrenoceptor agonists in such learning. The [beta]1-adrenoceptor agonist, dobutamine, and the [alpha]1-adrenoceptor agonist, phenylephrine, induced learning, and both exhibited an…

  9. Spontaneous multivessel cervical artery dissection in a patient with a substitution of alanine for glycine (G13A) in the alpha 1 (I) chain of type I collagen.

    PubMed

    Mayer, S A; Rubin, B S; Starman, B J; Byers, P H

    1996-08-01

    Cervical artery dissection occurs spontaneously and in multiple vessels with surprising frequency. An underlying arteriopathy is frequently suspected, but specific causes of vascular fragility are rarely identified. We describe a 35-year-old woman who developed multiple cervical artery dissections after scuba diving. She had no stigmata of connective tissue disease apart from bluish sclerae, and no family history of arterial dissection or congenital musculoskeletal disease. Analysis of the COL1A1 gene that encodes the pro alpha 1(I) chains of type I procollagen revealed a point mutation in one allele, resulting in substitution of alanine for glycine (G13A) in about half the alpha 1(I) chains of type I collagen. Genetic disorders of collagen, such as the mild phenotypic variant of osteogenesis imperfecta identified in our patient, should be considered in the differential diagnosis of unexplained cervical artery dissection.

  10. d-alpha-tocopherol inhibits collagen alpha 1(I) gene expression in cultured human fibroblasts. Modulation of constitutive collagen gene expression by lipid peroxidation.

    PubMed Central

    Houglum, K; Brenner, D A; Chojkier, M

    1991-01-01

    Ascorbic acid stimulates collagen gene transcription in cultured fibroblasts, and this effect is mediated through the induction of lipid peroxidation by ascorbic acid. Quiescent cultured fibroblasts in the absence of ascorbic acid have a high constitutive level of collagen production, but the mechanisms of collagen gene regulation in this unstimulated state are not known. Because lipid peroxidation also occurs in normal cells, we wondered if lipid peroxidation plays a role in the regulation of basal collagen gene expression. Inhibition of lipid peroxidation in cultured human fibroblasts with d-alpha-tocopherol or methylene blue decreased the synthesis of collagen, the steady-state levels of procollagen alpha 1(I) mRNA and the transcription of the procollagen alpha 1(I) gene. This effect on collagen gene expression was selective and not associated with cellular toxicity. Thus, these experiments suggest a role for lipid peroxidation in the modulation of constitutive collagen gene expression. Images PMID:2040703

  11. Preparation and Tungsten-183 NMR Characterization of [alpha-1-P(2)W(17)O(61)](10)(-), [alpha-1-Zn(H(2)O)P(2)W(17)O(61)](8)(-), and [alpha-2-Zn(H(2)O)P(2)W(17)O(61)](8)(-).

    PubMed

    Bartis, Judit; Kunina, Yuliya; Blumenstein, Michael; Francesconi, Lynn C.

    1996-03-13

    The preparation of the alpha-1 and alpha-2 isomers of the Wells-Dawson 17 tungsto derivatives by standard methods is accompanied by a significant proportion of the other isomer present as an impurity. In this study, the alpha-1 and alpha-2 isomers of [Zn(H(2)O)P(2)W(17)O(61)](8)(-) have been prepared in >98% purity by reacting isomerically pure K(9)Li[alpha-1-P(2)W(17)O(61)] and K(10)[alpha-2-P(2)W(17)O(61)], respectively, with ZnCl(2), while rigorously controlling the pH at 4.7. The molecules were isolated as potassium salts. For (183)W NMR and (31)P NMR characterization, both molecules were ion exchanged by cation-exchange chromatography, maintaining the pH at 4.7, to obtain the lithium salts. Removal of water and isolation of a solid sample of [alpha-1-Zn(H(2)O)P(2)W(17)O(61)](8)(-) was achieved by lyophilization at -40 degrees C. The chemical shift data from (31)P and (183)W NMR spectroscopy of the isolated [alpha-1-Zn(H(2)O)P(2)W(17)O(61)](8)(-) and [alpha-2-Zn(H(2)O)P(2)W(17)O(61)](8)(-) isomers are consistent with a mixture of the alpha-1 and alpha-2 isomers reported previously;(1) the molecules have the expected C(1) and C(s)() symmetry, respectively. The [alpha-1-Zn(H(2)O)P(2)W(17)O(61)](8)(-) isomer is stable in the pH range of 4.6-6 at temperatures <35 degrees C. Using the same ion exchange and lyophilization techniques, the lacunary [alpha-1-P(2)W(17)O(61)](10)(-) isomer was isolated as the lithium salt; characterization by (183)W NMR spectroscopy confirms the C(1) symmetry.

  12. Allergy to Red Meat: A Diagnosis Made by the Patient and Confirmed by an Assay for IgE Antibodies Specific for Alpha-1,3-Galactose.

    PubMed

    Kaloga, Mamadou; Kourouma, Sarah; Kouassi, Yao Isidore; Ecra, Elidje Joseph; Gbery, Ildevert Patrice; Allou, Ange S; Diabate, Almamy; Djeha, Djokouehi; Sangaré, Abdoulaye; Yoboue, Yao Pauline

    2016-01-01

    We report the first case of allergy to red meat observed in Ivory Coast. A 49-year-old male presented with pruritus. The diagnosis of allergy to red meat was confirmed by an assay for IgE antibodies specific for alpha-1,3 galactose. Interestingly, the disease was considered a spell to the patient who was suspected of being a sorcerer by the community. PMID:26933408

  13. Alpha-blockade therapy for benign prostatic hyperplasia: from a nonselective to a more selective alpha1A-adrenergic antagonist.

    PubMed

    Beduschi, M C; Beduschi, R; Oesterling, J E

    1998-06-01

    Benign prostatic hyperplasia (BPH) is very common in older men, causing symptoms that can markedly impair quality of life. Surgical treatment, typically transurethral resection of the prostate (TURP), is highly effective but can be costly and is associated with the risk for significant morbidity. Medical treatments for BPH are targeted toward reducing bladder outlet obstruction either by androgen blockade to reduce prostatic volume or alpha-adrenergic blockade to relax the smooth muscle tone of the prostate. In recent years, understanding of the sympathetic innervation of the prostate has improved. This has been paralleled by the development of alpha-adrenergic blocking agents, from nonselective alpha-antagonists, to selective alpha1-antagonists, to the more selective alpha1A-antagonists. It is anticipated that more specific agents will optimize the therapeutic effectiveness of alpha-adrenergic blockade in the prostate while reducing the side effects associated with alpha-adrenergic blockade in other areas of the body, such as the vascular system. This article reviews the evolution of alpha-blockade therapy in management of BPH, focusing on tamsulosin, an agent targeted toward the alpha1A-adrenoceptor that predominates in the prostate. Clinical trials in Europe and the United States have provided evidence that tamsulosin is effective at doses of 0.4 and 0.8 mg/day. At both doses, tamsulosin is associated with significant improvements in the American Urological Association symptom score and the mean and peak urinary flow rates as compared with placebo. This once-daily alpha1A-adrenergic antagonist is well-tolerated, with a minimal potential for the side effects associated with alphas-blocker therapy.

  14. The Structure and Function of an Arabinan-specific [alpha]-1,2-Arabinofuranosidase Identified from Screening the Activities of Bacterial GH43 Glycoside Hydrolases

    SciTech Connect

    Cartmell, Alan; McKee, Lauren S.; Pena, Maria J.; Larsbrink, Johan; Brumer, Harry; Kaneko, Satoshi; Ichinose, Hitomi; Lewis, Richard J.; Vikso-Nielsen, Anders; Gilbert, Harry; Marles-Wright, Jon

    2012-03-26

    Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo- and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific {alpha}-1,2-arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave {alpha}-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific {alpha}-1,2-arabinofuranosidase, CjAbf43A, displays a five-bladed {beta}-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for {alpha}-1,2-L-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.

  15. Identification of a GH110 subfamily of alpha 1,3-galactosidases: novel enzymes for removal of the alpha 3Gal xenotransplantation antigen.

    PubMed

    Liu, Qiyong P; Yuan, Huaiping; Bennett, Eric P; Levery, Steven B; Nudelman, Edward; Spence, Jean; Pietz, Greg; Saunders, Kristen; White, Thayer; Olsson, Martin L; Henrissat, Bernard; Sulzenbacher, Gerlind; Clausen, Henrik

    2008-03-28

    In search of alpha-galactosidases with improved kinetic properties for removal of the immunodominant alpha1,3-linked galactose residues of blood group B antigens, we recently identified a novel prokaryotic family of alpha-galactosidases (CAZy GH110) with highly restricted substrate specificity and neutral pH optimum (Liu, Q. P., Sulzenbacher, G., Yuan, H., Bennett, E. P., Pietz, G., Saunders, K., Spence, J., Nudelman, E., Levery, S. B., White, T., Neveu, J. M., Lane, W. S., Bourne, Y., Olsson, M. L., Henrissat, B., and Clausen, H. (2007) Nat. Biotechnol. 25, 454-464). One member of this family from Bacteroides fragilis had exquisite substrate specificity for the branched blood group B structure Galalpha1-3(Fucalpha1-2)Gal, whereas linear oligosaccharides terminated by alpha1,3-linked galactose such as the immunodominant xenotransplantation epitope Galalpha1-3Galbeta1-4GlcNAc did not serve as substrates. Here we demonstrate the existence of two distinct subfamilies of GH110 in B. fragilis and thetaiotaomicron strains. Members of one subfamily have exclusive specificity for the branched blood group B structures, whereas members of a newly identified subfamily represent linkage specific alpha1,3-galactosidases that act equally well on both branched blood group B and linear alpha1,3Gal structures. We determined by one-dimensional (1)H NMR spectroscopy that GH110 enzymes function with an inverting mechanism, which is in striking contrast to all other known alpha-galactosidases that use a retaining mechanism. The novel GH110 subfamily offers enzymes with highly improved performance in enzymatic removal of the immunodominant alpha3Gal xenotransplantation epitope.

  16. Synthesis and alpha 1-adrenoceptor antagonistic activity of some 4-amino-5,7-dimethyl-2-(substituted) aminopyrido (2,3-d) pyrimidines.

    PubMed

    Chhabria, Mahesh T; Srinivas, Sreeramaiah; Rajan, Kombu S; Ravikumar, Tadiparthy; Rathnam, Shivprakash

    2002-01-01

    A series of new 4-amino-5,7-dimethyl-2- (substituted)aminopyrido(2,3-d)pyrimidines (5) have been synthesized and tested for selective alpha 1-adrenoreceptor antagonistic activity. Some of the compounds were found to antagonize alpha 1-adrenoreceptor in a competitive and reversible manner. When screened on rat anococcygeus muscle some of the compounds exhibited significant alpha 1-adrenoreceptor antagonistic activity (pA2 values in the range of 5.2-7.8). The most potent compound (5j) was evaluated by an in vivo method and was found to reduce the systolic and diastolic blood pressure of spontaneously hypertensive rats. The percentage reduction in blood pressure by test compound 5j was found to be higher than that of the standard drug prazosin (CAS 19216-56-9) at the same dose level (1 mg/kg p.o.). Chemically, prazosin is a 4-aminoquinazolin derivative. Pyridopyrimidine is a known bioisostere of quinazoline. The study revealed that isosteric replacement of the benzene ring of prazosin by a pyridine ring increases the potency.

  17. Involvement of dopamine D1 receptors and alpha1-adrenoceptors in the antidepressant-like effect of chlorpheniramine in the mouse tail suspension test.

    PubMed

    Hirano, Shoko; Miyata, Shigeo; Onodera, Kenji; Kamei, Junzo

    2007-05-01

    It has been reported that chlorpheniramine, a classical antihistamine, has antidepressant-like effects in animal models of depression. In this study, we examined the involvement of dopaminergic (dopamine D(1) and dopamine D(2) receptors), noradrenergic (alpha(1)- and beta-adrenoceptors) and serotonergic (5-HT(1A) and 5-HT(2) receptors) receptors in the antidepressant-like effect of chlorpheniramine in the mouse tail suspension test. We also investigated the involvement of these monoamine receptors in the antidepressant-like effect of imipramine for comparison with the mechanisms of the effect of chlorpheniramine. Both imipramine and chlorpheniramine significantly reduced the duration of immobility in the tail suspension test without affecting spontaneous locomotor activity in mice. The anti-immobility effect of imipramine (30 mg/kg, i.p.) was significantly antagonized by the selective dopamine D(1) receptor antagonist SCH23390 but not by the other receptor antagonists. In contrast, the anti-immobility effect of chlorpheniramine was significantly inhibited by SCH23390 and the selective alpha(1)-adrenoceptor antagonist prazosin, but not by the other receptor antagonists. In conclusion, these results suggest that chlorpheniramine exerts an antidepressant-like effect in the mouse tail suspension test that is mediated by at least the activation of dopamine D(1) receptors and alpha(1)-adrenoceptors. In addition, the antidepressant-like effect of chlorpheniramine may be induced by several mechanisms that are different from those involved in the antidepressant-like effect of imipramine.

  18. Essential, completely conserved glycine residue in the domain III S2-S3 linker of voltage-gated calcium channel alpha1 subunits in yeast and mammals.

    PubMed

    Iida, Kazuko; Teng, Jinfeng; Tada, Tomoko; Saka, Ayaka; Tamai, Masumi; Izumi-Nakaseko, Hiroko; Adachi-Akahane, Satomi; Iida, Hidetoshi

    2007-08-31

    Voltage-gated Ca2+ channels (VGCCs) mediate the influx of Ca2+ that regulates many cellular events, and mutations in VGCC genes cause serious hereditary diseases in mammals. The yeast Saccharomyces cerevisiae has only one gene encoding the putative pore-forming alpha1 subunit of VGCC, CCH1. Here, we identify a cch1 allele producing a completely nonfunctional Cch1 protein with a Gly1265 to Glu substitution present in the domain III S2-S3 cytoplasmic linker. Comparison of amino acid sequences of this linker among 58 VGCC alpha1 subunits from 17 species reveals that a Gly residue whose position corresponds to that of the Cch1 Gly1265 is completely conserved from yeasts to humans. Systematic amino acid substitution analysis using 10 amino acids with different chemical and structural properties indicates that the Gly1265 is essential for Cch1 function because of the smallest residue volume. Replacement of the Gly959 residue of a rat brain Cav1.2 alpha1 subunit (rbCII), positionally corresponding to the yeast Cch1 Gly1265, with Glu, Ser, Lys, or Ala results in the loss of Ba2+ currents, as revealed by the patch clamp method. These results suggest that the Gly residue in the domain III S2-S3 linker is functionally indispensable from yeasts to mammals. Because the Gly residue has never been studied in any VGCC, these findings provide new insights into the structure-function relationships of VGCCs.

  19. Design and synthesis of novel alpha(1)(a) adrenoceptor-selective antagonists. 2. Approaches to eliminate opioid agonist metabolites via modification of linker and 4-methoxycarbonyl-4-phenylpiperidine moiety.

    PubMed

    Murali Dhar, T G; Nagarathnam, D; Marzabadi, M R; Lagu, B; Wong, W C; Chiu, G; Tyagarajan, S; Miao, S W; Zhang, F; Sun, W; Tian, D; Shen, Q; Zhang, J; Wetzel, J M; Forray, C; Chang, R S; Broten, T P; Schorn, T W; Chen, T B; O'Malley, S; Ransom, R; Schneck, K; Bendesky, R; Harrell, C M; Vyas, K P

    1999-11-18

    We have previously described compound 1a as a high-affinity subtype selective alpha(1a) antagonist. In vitro and in vivo evaluation of compound 1a showed its major metabolite to be a mu-opioid agonist, 4-methoxycarbonyl-4-phenylpiperidine (3). Several dihydropyrimidinone analogues were synthesized with the goal of either minimizing the formation of 3 by modification of the linker or finding alternative piperidine moieties which when cleaved as a consequence of metabolism would not give rise to mu-opioid activity. Modification of the linker gave several compounds with good alpha(1a) binding affinity (K(i) = < 1 nM) and selectivity (>300-fold over alpha(1b) and alpha(1d)). In vitro analysis in the microsomal assay revealed these modifications did not significantly affect N-dealkylation and the formation of the piperidine 3. The second approach, however, yielded several piperidine replacements for 3, which did not show significant mu-opioid activity. Several of these compounds maintained good affinity at the alpha(1a) adrenoceptor and selectivity over alpha(1b) and alpha(1d). For example, the piperidine fragments of (+)-73 and (+)-83, viz. 4-cyano-4-phenylpiperidine and 4-methyl-4-phenylpiperidine, were essentially inactive at the mu-opioid receptor (IC(50) > 30 microM vs 3 microM for 3). Compounds (+)-73 and (+)-83 were subjected to detailed in vitro and in vivo characterization. Both these compounds, in addition to their excellent selectivity (>880-fold) over alpha(1b) and alpha(1d), also showed good selectivity over several other recombinant human G-protein coupled receptors. Compounds (+)-73 and (+)-83 showed good functional potency in isolated human prostate tissues, with K(b)s comparable to their in vitro alpha(1a) binding data. In addition, compound (+)-73 also exhibited good uroselectivity (DBP K(b)/IUP K(b) > 20-fold) in the in vivo experiments in dogs, similar to 1a.

  20. Inhibition of human ether-a-go-go-related gene potassium channels by alpha 1-adrenoceptor antagonists prazosin, doxazosin, and terazosin.

    PubMed

    Thomas, Dierk; Wimmer, Anna-Britt; Wu, Kezhong; Hammerling, Bettina C; Ficker, Eckhard K; Kuryshev, Yuri A; Kiehn, Johann; Katus, Hugo A; Schoels, Wolfgang; Karle, Christoph A

    2004-05-01

    Human ether-a-go-go-related gene (HERG) potassium channels are expressed in multiple tissues including the heart and adenocarcinomas. In cardiomyocytes, HERG encodes the alpha-subunit underlying the rapid component of the delayed rectifier potassium current, I(Kr), and pharmacological reduction of HERG currents may cause acquired long QT syndrome. In addition, HERG currents have been shown to be involved in the regulation of cell proliferation and apoptosis. Selective alpha 1-adrenoceptor antagonists are commonly used in the treatment of hypertension and benign prostatic hyperplasia. Recently, doxazosin has been associated with an increased risk of heart failure. Moreover, quinazoline-derived alpha 1-inhibitors induce apoptosis in cardiomyocytes and prostate tumor cells independently of alpha1-adrenoceptor blockade. To assess the action of the effects of prazosin, doxazosin, and terazosin on HERG currents, we investigated their acute electrophysiological effects on cloned HERG potassium channels heterologously expressed in Xenopus oocytes and HEK 293 cells.Prazosin, doxazosin, and terazosin blocked HERG currents in Xenopus oocytes with IC(50) values of 10.1, 18.2, and 113.2 microM respectively, whereas the IC(50) values for HERG channel inhibition in human HEK 293 cells were 1.57 microM, 585.1 nM, and 17.7 microM. Detailed biophysical studies revealed that inhibition by the prototype alpha 1-blocker prazosin occurred in closed, open, and inactivated channels. Analysis of the voltage-dependence of block displayed a reduction of inhibition at positive membrane potentials. Frequency-dependence was not observed. Prazosin caused a negative shift in the voltage-dependence of both activation (-3.8 mV) and inactivation (-9.4 mV). The S6 mutations Y652A and F656A partially attenuated (Y652A) or abolished (F656A) HERG current blockade, indicating that prazosin binds to a common drug receptor within the pore-S6 region. In conclusion, this study demonstrates that HERG

  1. Agonist-dependent single channel current and gating in alpha4beta2delta and alpha1beta2gamma2S GABAA receptors.

    PubMed

    Keramidas, Angelo; Harrison, Neil L

    2008-02-01

    The family of gamma-aminobutyric acid type A receptors (GABA(A)Rs) mediates two types of inhibition in the mammalian brain. Phasic inhibition is mediated by synaptic GABA(A)Rs that are mainly comprised of alpha(1), beta(2), and gamma(2) subunits, whereas tonic inhibition is mediated by extrasynaptic GABA(A)Rs comprised of alpha(4/6), beta(2), and delta subunits. We investigated the activation properties of recombinant alpha(4)beta(2)delta and alpha(1)beta(2)gamma(2S) GABA(A)Rs in response to GABA and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one (THIP) using electrophysiological recordings from outside-out membrane patches. Rapid agonist application experiments indicated that THIP produced faster opening rates at alpha(4)beta(2)delta GABA(A)Rs (beta approximately 1600 s(-1)) than at alpha(1)beta(2)gamma(2S) GABA(A)Rs (beta approximately 460 s(-1)), whereas GABA activated alpha(1)beta(2)gamma(2S) GABA(A)Rs more rapidly (beta approximately 1800 s(-1)) than alpha(4)beta(2)delta GABA(A)Rs (beta < 440 s(-1)). Single channel recordings of alpha(1)beta(2)gamma(2S) and alpha(4)beta(2)delta GABA(A)Rs showed that both channels open to a main conductance state of approximately 25 pS at -70 mV when activated by GABA and low concentrations of THIP, whereas saturating concentrations of THIP elicited approximately 36 pS openings at both channels. Saturating concentrations of GABA elicited brief (<10 ms) openings with low intraburst open probability (P(O) approximately 0.3) at alpha(4)beta(2)delta GABA(A)Rs and at least two "modes" of single channel bursting activity, lasting approximately 100 ms at alpha(1)beta(2)gamma(2S) GABA(A)Rs. The most prevalent bursting mode had a P(O) of approximately 0.7 and was described by a reaction scheme with three open and three shut states, whereas the "high" P(O) mode ( approximately 0.9) was characterized by two shut and three open states. Single channel activity elicited by THIP in alpha(4)beta(2)delta and alpha(1)beta(2)gamma(2S) GABA

  2. Diagnosis of. alpha. sub 1 -antitrypsin deficiency by enzymatic amplification of human genomic DNA and direct sequencing of polymerase chain reaction products

    SciTech Connect

    Newton, C.R.; Graham, A.; Powell, S.; Gammack, A.; Riley, J.; Markham, A.F. ); Kalsheker, N. )

    1988-09-12

    The authors have compared sequencing of cloned polymerase chain reaction (PCR) products and the direct sequencing of PCR products in the examination of individuals from six families affected with {alpha}{sub 1}-antitrypsin (AAT) deficiency. In families where paternity was in question they confirmed consanguinity by DNA fingerprinting using a panel of locus-specific minisatellite probes. They demonstrate that direct sequencing of PCR amplification products is the method of choice for the absolutely specific diagnosis of AAT deficiency and can distinguish normals, heterozygotes and homozygotes in a single, rapid and facile assay. Furthermore, they demonstrate the reproducibility of the PCR and a rapid DNA isolation procedure. They have also shown that two loci can be simultaneously amplified and that the PCR product from each locus can be independently examined by direct DNA sequencing.

  3. The Shapes of Z-α1-Antitrypsin Polymers in Solution Support the C-Terminal Domain-Swap Mechanism of Polymerization

    PubMed Central

    Behrens, Manja A.; Sendall, Timothy J.; Pedersen, Jan S.; Kjeldgaard, Morten; Huntington, James A.; Jensen, Jan K.

    2014-01-01

    Emphysema and liver cirrhosis can be caused by the Z mutation (Glu342Lys) in the serine protease inhibitor α1-antitrypsin (α1AT), which is found in more than 4% of the Northern European population. Homozygotes experience deficiency in the lung concomitantly with a massive accumulation of polymers within hepatocytes, causing their destruction. Recently, it was proposed that Z-α1AT polymerizes by a C-terminal domain swap. In this study, small-angle x-ray scattering (SAXS) was used to characterize Z-α1AT polymers in solution. The data show that the Z-α1AT trimer, tetramer, and pentamer all form ring-like structures in strong support of a common domain-swap polymerization mechanism that can lead to self-terminating polymers. PMID:25418171

  4. The shapes of Z-α1-antitrypsin polymers in solution support the C-terminal domain-swap mechanism of polymerization.

    PubMed

    Behrens, Manja A; Sendall, Timothy J; Pedersen, Jan S; Kjeldgaard, Morten; Huntington, James A; Jensen, Jan K

    2014-10-21

    Emphysema and liver cirrhosis can be caused by the Z mutation (Glu342Lys) in the serine protease inhibitor α1-antitrypsin (α1AT), which is found in more than 4% of the Northern European population. Homozygotes experience deficiency in the lung concomitantly with a massive accumulation of polymers within hepatocytes, causing their destruction. Recently, it was proposed that Z-α1AT polymerizes by a C-terminal domain swap. In this study, small-angle x-ray scattering (SAXS) was used to characterize Z-α1AT polymers in solution. The data show that the Z-α1AT trimer, tetramer, and pentamer all form ring-like structures in strong support of a common domain-swap polymerization mechanism that can lead to self-terminating polymers.