Regulation of folate reductase synthesis in sensitive and methotrexate-resistant sarcoma 180 cells. In vitro translation and characterization of folate reductase mRNA.

A highly specific assay for folate reductase mRNA activity from Sarcoma 180 cells was developed using the rabbit reticulocyte lysate protein synthesizing system. Quantitation of in vitro folate reductase synthesis was accomplished by direct immunoprecipitation from lysate reactions. The in vitro labeled folate reductase was synthesized in a linear response to a wide range of RNA concentrations, migrated as a single prominent radioactive species upon polyacrylamide gel electrophoresis, and was indistinguishable from authentic 14C-labeled folate reductase on the basis of molecular weight and immunotitration with anti-folate reductase gamma-globulin. The assay was used to quantitate folate reductase mRNA activity in various cell lines and under several conditions known to affect folate reductase synthesis. These included (a) sensitive and methotrexate-resistant Sarcoma 180 cells, (b) two lines of resistant cells having different relative rates of folate reductase synthesis, (c) growth of methotrexate-resistant cells in the absence of methotrexate, and (d) growth phase. The results indicate that the relative rate of folate reductase synthesis in each case can be explained solely by the level of translatable folate reductase mRNA. The use of poly(U)-Sepharose and sucrose gradient fractionation procedures indicated that folate reductase mRNA contains poly(A) and has a sedimentation coefficient of approximately 14 S. These two fractionation steps were combined to achieve an approximately 90-fold purification of folate reductase mRNA over total cytoplasmic RNA.     

Differences in growth and water relations among Phaseolus vulgaris cultivars in response to induced drought stress

Relatively little ecophysiological research has been conducted to determine the responses to drought of Phaseolus vulgaris. Four bean cultivars (cvs.) from Brazil, A320, Carioca, Ouro Negro and Xodó were submitted to an imposed water deficit in order to evaluate the importance of some adaptive mechanisms of drought resistance through the analysis of growth parameters, water status, gas exchange and indicators of tolerance mechanisms at the cellular level. During the drought treatment, relative growth rates were more reduced for A320 and Xodó than Carioca and Ouro Negro. A320 closed its stomata very rapidly and complete stomatal closure was obtained at Psi(w)=-0.6 MPa, in contrast to the other cvs. where stomata were fully closed only at Psi(w)=-0.9 MPa. Net assimilation rates were closely related to stomatal conductances. Mechanisms at the cellular level appeared to be mostly important for higher tolerance. Carioca and Ouro Negro, when compared to A320 and Xodó, were characterized by having better drought tolerance mechanisms and higher tissue water retention capacity leading to a better growth under water deficits. The leaf dehydration rates of those cvs. were slow whereas those of the drought sensitive cvs. were rapid. The results were confirmed by the electrolyte leakage test and leaf osmotic potential measurements, which indicated higher membrane resistance and osmotic adjustment in the two tolerant cvs. Carioca and Ouro Negro. It appears from this study that despite being cultivated in the same geographical region, the four cvs. of P. vulgaris displayed somewhat different drought adaptive capacities for prolonged drought during the vegetative phase.     

Adenosine-dependent pulmonary fibrosis in adenosine deaminase-deficient mice

Pulmonary fibrosis is a common feature of numerous lung disorders, including interstitial lung diseases, asthma, and chronic obstructive pulmonary disease. Despite the prevalence of pulmonary fibrosis, the molecular mechanisms governing inflammatory and fibroproliferative aspects of the disorder are not clear. Adenosine is a purine-signaling nucleoside that is generated in excess during cellular stress and damage. This signaling molecule has been implicated in the regulation of features of chronic lung disease; however, the impact of adenosine on pulmonary fibrosis is not well understood. The goal of this study was to explore the impact of endogenous adenosine elevations on pulmonary fibrosis. To accomplish this, adenosine deaminase (ADA)-deficient mice were treated with various levels of ADA enzyme replacement therapy to regulate endogenous adenosine levels in the lung. Maintaining ADA-deficient mice on low dosages of ADA enzyme therapy led to chronic elevations in lung adenosine levels that were associated with pulmonary inflammation, expression of profibrotic molecules, collagen deposition, and extreme alteration in airway structure. These features could be blocked by preventing elevations in lung adenosine. Furthermore, lowering lung adenosine levels after the establishment of pulmonary fibrosis resulted in a resolution of fibrosis. These findings demonstrate that chronic adenosine elevations are associated with pulmonary fibrosis in ADA-deficient mice and suggest that the adenosine functions as a profibrotic signal in the lung.     

The importance of adenosine deaminase for lymphocyte development and function

Deficiency in the enzyme adenosine deaminase (ADA) in humans manifests primarily as severe lymphopenia and immunodeficiency, resulting in death by 6 months of age, if untreated. In this review, we discuss phenotypical, biochemical, and metabolic hallmarks of the disease, and describe a mouse model in which levels of ADA can be biochemically and genetically manipulated. This model provides exciting possibilities for uncovering the mechanisms by which this purine catabolic enzyme affects lymphopoiesis.     

Reduced N cycling in response to elevated CO2, warming,and drought in a Danish heathland: Synthesizing results of the CLIMAITE project after two years of treatments

Field‐scale experiments simulating realistic future climate scenarios are important tools for investigating the effects of current and future climate changes on ecosystem functioning and biogeochemical cycling. We exposed a seminatural Danish heathland ecosystem to elevated atmospheric carbon dioxide (CO₂), warming, and extended summer drought in all combinations. Here, we report on the short‐term responses of the nitrogen (N) cycle after 2 years of treatments. Elevated CO₂ significantly affected aboveground stoichiometry by increasing the carbon to nitrogen (C/N) ratios in the leaves of both co‐dominant species (Calluna vulgaris and Deschampsia flexuosa), as well as the C/N ratios of Calluna flowers and by reducing the N concentration of Deschampsia litter. Belowground, elevated CO₂ had only minor effects, whereas warming increased N turnover, as indicated by increased rates of microbial NH₄⁺ consumption, gross mineralization, potential nitrification, denitrification and N₂O emissions. Drought reduced belowground gross N mineralization and decreased fauna N mass and fauna N mineralization. Leaching was unaffected by treatments but was significantly higher across all treatments in the second year than in the much drier first year indicating that ecosystem N loss is highly sensitive to changes and variability in amount and timing of precipitation. Interactions between treatments were common and although some synergistic effects were observed, antagonism dominated the interactive responses in treatment combinations, i.e. responses were smaller in combinations than in single treatments. Nonetheless, increased C/N ratios of photosynthetic tissue in response to elevated CO₂, as well as drought‐induced decreases in litter N production and fauna N mineralization prevailed in the full treatment combination. Overall, the simulated future climate scenario therefore lead to reduced N turnover, which could act to reduce the potential growth response of plants to elevated atmospheric CO₂ concentration.     

Autoantibody-mediated IL-6-dependent endothelin-1 elevation underlies pathogenesis in a mouse model of preeclampsia

Preeclampsia (PE) is a life-threatening hypertensive disorder of pregnancy. Elevated circulating endothelin-1 (ET-1) is associated with the disease. However the molecular basis of increased ET-1 production and its role in PE are unknown. This study aimed to investigate the causative factors, pathological role of elevated ET-1 production in PE, and the underlying mechanisms. In this study, we found that IgG from women with PE, in contrast to IgG from normotensive pregnant women, induced preproET-1 mRNA expression via angiotensin II type 1 receptor activation in kidneys and placentas in pregnant mice. The ET-A receptor-specific antagonist BQ123 significantly attenuated autoantibody-induced hypertension, proteinuria, and renal damage in pregnant mice, demonstrating that autoantibody-induced ET-1 production contributes to pathophysiology. Mechanistically, we discovered that IL-6 functioned downstream of TNF-α signaling, contributing to increased ET-1 production in pregnant mice. IL-6 blockade inhibited preeclamptic features in autoantibody-injected pregnant mice. Extending the data to human studies, we found that IL-6 was a key cytokine underlying ET-1 induction mediated by IgG from women with PE in human placental villous explants and that endothelial cells are a key source of ET-1. Overall, we provide human and mouse studies showing that angiotensin II type I receptor-agonistic autoantibody is a novel causative factor responsible for elevated ET-1 production and that increased TNF-α/IL-6 signaling is a key mechanism underlying increased ET-1 production and subsequent maternal features. Significantly, our findings revealed novel factors and signaling cascades involved in ET-1 production, subsequent disease symptom development, and possible therapeutic intervention in the management of PE.     

Angiotensin receptors, autoimmunity, and preeclampsia

Preeclampsia is a pregnancy-induced hypertensive disorder that causes substantial maternal and fetal morbidity and mortality. Despite being a leading cause of maternal death and a major contributor to maternal and perinatal morbidity, the mechanisms responsible for the pathogenesis of preeclampsia are poorly understood. Recent studies indicate that women with preeclampsia have autoantibodies that activate the angiotensin receptor, AT1, and that autoantibody-mediated receptor activation contributes to pathophysiology associated with preeclampsia. The research reviewed here raises the intriguing possibility that preeclampsia may be a pregnancy-induced autoimmune disease.     

Human immunodeficiency virus seroconversion presenting with acute inflammatory demyelinating polyneuropathy: a case report

Introduction

Electrocardiogram (ECG) abnormalities in patients with blunt chest trauma are diverse and non-specific, but may be indicative of potentially life-threatening conditions.

Case presentation

We report a rare case of pneumopericardium with extreme ECG abnormalities after blunt chest trauma in a 22-year-old male. The diagnosis was confirmed using computed tomography (CT) scanning. The case is discussed, together with its differential diagnosis and the aetiology of pneumopericardium and tension pneumopericardium.

Conclusion

Pneumopericardium should be distinguished from other pathologies such as myocardial contusion and myocardial infarction because of the possible development of tension pneumopericardium. Early CT scanning is important in the evaluation of blunt chest trauma.     

Control of cellular gene expression during adenovirus infection: induction and shut-off of dihydrofolate reductase gene expression by adenovirus type 2.

Infection of human cells by adenovirus results in multiple alterations of host gene expression. To examine the effects of viral infection on the expression of a single gene, a line of human cells was developed which is resistant to growth in methotrexate and which contains amplified RNA and protein specific for dihydrofolate reductase (DHFR). Cytogenetic evidence indicated the presence of amplified DNA. Adenovirus infection of these cells caused an induction and subsequent decline in the synthesis of DHFR protein. The maximum DHFR induction occurred 16 to 19 h after infection and reached a level 2.5-fold greater than that observed in uninfected cells. Induction of DHFR protein synthesis was accompanied by concomitant increases in the level of steady-state DHFR-specific cytoplasmic RNA. The relative rate of DHFR mRNA production (i.e., the appearance of DHFR-specific mRNA sequences in the cytoplasm) also increased 2.5-fold during induction. Later in infection, the relative rate of DHFR protein synthesis declined, reaching a level below that observed in uninfected cells. This decline was accompanied by a similar decline in the steady-state levels of DHFR RNA and in the relative rate of synthesis of DHFR mRNA. These data suggest that adenovirus infection controls DHFR gene expression by increasing and subsequently decreasing the relative rate at which DHFR-specific mRNA sequences appear in the cytoplasm and enter the pool of mRNA available for translation.     

Deduced amino acid sequence of Escherichia coli adenosine deaminase reveals evolutionarily conserved amino acid residues: implications for catalytic function

The goal of the research reported here is to identify evolutionarily conserved amino acid residues associated with enzymatic deamination of adenosine. To do this, we isolated molecular clones of the Escherichia coli adenosine deaminase gene by functional complementation of adenosine deaminase deficient bacteria and deduced the amino acid sequence of the enzyme from the nucleotide sequence of the gene. Nucleotide sequence analysis revealed the presence of a 996-nucleotide open reading frame encoding a protein of 332 amino acids having a molecular weight of 36,345. The deduced amino acid sequence of the E. coli enzyme has approximately 33% identity with those of the mammalian adenosine deaminases. With conservative amino acid substitutions the overall sequence homology approaches 50%, suggesting that the structures and functions of the mammalian and bacterial enzymes are similar. Additional amino acid sequence analysis revealed specific residues that are conserved among all three adenosine deaminases and four AMP deaminases for which sequence information is currently available. In view of previously published enzymological data and the conserved amino acid residues identified in this study, we propose a model to account for the enzyme-catalyzed hydrolytic deamination of adenosine. Potential catalytic roles are assigned to the conserved His 214, Cys 262, Asp 295, and Asp 296 residues of mammalian adenosine deaminases and the corresponding conserved amino acid residues in bacterial adenosine deaminase and the eukaryotic AMP deaminases.