High CO2 levels impair alveolar epithelial function independently of pH

Background

In patients with acute respiratory failure, gas exchange is impaired due to the accumulation of fluid in the lung airspaces. This life-threatening syndrome is treated with mechanical ventilation, which is adjusted to maintain gas exchange, but can be associated with the accumulation of carbon dioxide in the lung. Carbon dioxide (CO₂) is a by-product of cellular energy utilization and its elimination is affected via alveolar epithelial cells. Signaling pathways sensitive to changes in CO₂ levels were described in plants and neuronal mammalian cells. However, it has not been fully elucidated whether non-neuronal cells sense and respond to CO₂. The Na,K-ATPase consumes ∼40% of the cellular metabolism to maintain cell homeostasis. Our study examines the effects of increased pCO₂ on the epithelial Na,K-ATPase a major contributor to alveolar fluid reabsorption which is a marker of alveolar epithelial function.

Principal Findings

We found that short-term increases in pCO₂ impaired alveolar fluid reabsorption in rats. Also, we provide evidence that non-excitable, alveolar epithelial cells sense and respond to high levels of CO₂, independently of extracellular and intracellular pH, by inhibiting Na,K-ATPase function, via activation of PKCζ which phosphorylates the Na,K-ATPase, causing it to endocytose from the plasma membrane into intracellular pools.

Conclusions

Our data suggest that alveolar epithelial cells, through which CO₂ is eliminated in mammals, are highly sensitive to hypercapnia. Elevated CO₂ levels impair alveolar epithelial function, independently of pH, which is relevant in patients with lung diseases and altered alveolar gas exchange.     

Topoisomerase IIIalpha is required for normal proliferation and telomere stability in alternative lengthening of telomeres

Topoisomerase (Topo) IIIalpha associates with BLM helicase, which is proposed to be important in the alternative lengthening of telomeres (ALT) pathway that allows telomere recombination in the absence of telomerase. Here, we show that human Topo IIIalpha colocalizes with telomeric proteins at ALT-associated promyelocytic bodies from ALT cells. In these cells, Topo IIIalpha immunoprecipitated with telomere binding protein (TRF) 2 and BLM and was shown to be associated with telomeric DNA by chromatin immunoprecipitation, suggesting that these proteins form a complex at telomere sequences. Topo IIIalpha depletion by small interfering RNA reduced ALT cell survival, but did not affect telomerase-positive cell lines. Moreover, repression of Topo IIIalpha expression in ALT cells reduced the levels of TRF2 and BLM proteins, provoked a strong increase in the formation of anaphase bridges, induced the degradation of the G-overhang signal, and resulted in the appearance of DNA damage at telomeres. In contrast, telomere maintenance and TRF2 levels were unaffected in telomerase-positive cells. We conclude that Topo IIIalpha is an important telomere-associated factor, essential for telomere maintenance and chromosome stability in ALT cells, and speculate on its potential mechanistic function.     

Myocardial hypertrophy in rats exposed to simulated high altitude

Myocardial hypertrophy in Sprague-Dawley adult rats exposed to hypobaric hypoxia (0.40 atmosphere of air/18 h daily for 7 days) in a hypobaric chamber was investigated. Changes in the myocardial mass were evaluated on the basis of the dry heart weight and expressed as mg/100 g of total body weight (mean +/- SEM). Data are presented indicating that: chronic hypobaric hypoxia causes a significant degree of myocardial hypertrophy in rats; hypertrophic process involves both ventricles (the right more than the left); removal of the hypoxic stimulus leads to the disappearance of hypertrophy when evaluated as an increase in dry heart weight; hypoxia affects the synthesis of a significant amount of connective tissue in the left ventricle, which is not exposed to pressure load. The r?le of neurohumoral factors (i.e., adrenergic stimulation and catecholamines) in the development of the ventricular hypertrophy is suggested.     

Entamoeba histolytica uses ferritin as an iron source and internalises this protein by means of clathrin-coated vesicles

Entamoeba histolytica is a parasitic protozoan that produces dysentery and often reaches the liver, leading to abscess formation. Ferritin is an iron-storage protein that is mainly found in liver and spleen in mammals. The liver contains a plentiful source of iron for amoebae multiplying in that organ, making it a prime target for infection since iron is essential for the growth of this parasite. The aim of this study was to determine whether trophozoites are able to take up ferritin and internalise this protein for their growth in axenic culture. Interaction between the amoebae and ferritin was studied by flow cytometry, confocal laser-scanning microscopy and transmission electron microscopy. Amoebae were viable in iron supplied by ferritin. Trophozoites quickly internalised ferritin via clathrin-coated vesicles, a process that was initiated within the first 2 min of incubation. In 30 min, ferritin was found colocalizing with the LAMP-2 protein at vesicles in the cytosol. The uptake of ferritin was time- temperature- and concentration-dependent, specific and saturated at 46 nM of ferritin. Haemoglobin and holo-transferrin did not compete with ferritin for binding to amoebae. Amoebae cleaved ferritin leading to the production of several different sized fragments. Cysteine proteases of 100, 75 and 50 kDa from amoeba extracts were observed in gels copolymerised with ferritin. For a pathogen such as E. histolytica, the capacity to utilise ferritin as an iron source may well explain its high pathogenic potential in the liver.     

pH control of limonin debittering with entrapped Rhodococcus fascians cells

Summary Rhodococcus fascians cells were immobilized by entrapment in -carrageenan. The ability of the system to continuously degrade limonin was tested against pH. A burst of activity was observed when changing from pH 4.5 to 5.0, and a small increase could be seen above the latter value. Such behaviour was not only a response of the metabolic activity of the cells to changes in the medium pH, but to selectivity towards the chemical form of the limonin substrate, which also depends on pH. Additionally, the immobilized cells showed increased resistance against pH changes, since the system recovered almost full activity when the pH was restored to 7.0 after being operated for long periods at pH 4.0. The decrease in limonin-degrading capability of the immobilized cells at low pH values could be overcome by choosing an appropriate dilution rate.Offprint requests to: J. L. Iborra     

On the identity of dnaP and dnaF genes of Bacillus subtilis

Summary The dnaP strains of Bacillus subtilis are altered in the initiation of DNA replication at high temperature (Riva et al., 1975). Fine mapping of the gene shows that it is located very close to the dnaF gene, described by Karamata and Gross (1970) and mapped by Love et al. (1976) in the polC region. The phenotype of both mutants is indistinguishable: the DNA synthesis stops at non permissive temperature after synthesizing an amount of DNA equivalent to the completion of the rounds of replication already initiated; at permissive temperature they are abnormally sensitive to MMS and are reduced in the ability to be transformed. Both mutants are to be considered as belonging to the dnaF locus.The dnaF gene is very close to the polC gene, which specifies the DNA polymerase III of B. subtilis. The DNA polymerase III of the dnaF mutants is not temperature sensitive in vitro, however, the level of this enzyme is lower by a factor of 4 or 5 in the dnaF mutants, at the permissive temperature. Following shift of dnaF cultures to the non permissive temperature, the level of DNA polymerase III activity specifically decreases further by a factor of at least 10 in the mutant, whereas the DNA polymerase I level is unaffected.The possible roles of the dnaF gene in the control of the cellular level of the DNA polymerase III, and the possibility of a regulatory role of DNA polymerase III in the initiation of DNA replication in bacteria are discussed.Abbreviations and symbols HPUra 6-(p-hydroxyphenylazo)-uracil; mic, minimum inhibitory concentration - MMS methyl-methanesufonate - Pol I Pol II and Pol III: DNA polymerase I, II and III respectively - PCMB parachloro-mercuri-benzoate     

Identification of Endothelin Receptor Subtype (ETB) in Human Cerebral Cortex Using Subtype-Selective Ligands

Abstract: Specific endothelin (ET) binding sites were characterized in membranes prepared from human cerebral cortices using binding assay and cross-linking analysis. The presence of immunoreactive (IR) ET-1 was studied by radioimmunoassay. Saturation binding experiments revealed that the K _D and B _max for ¹²⁵I-ET-1 and ¹²⁵l-ET-3 to membranes from gray matter were 25 ± 6 pM and 115 ± 15 fmol/mg of protein and 24 ± 5 p M and 108 ± 13 fmol/mg of protein, respectively. Similar results were obtained for white matter. In the presence of 10 n M sarafotoxin-6c, which is selective for ET_B receptors, ¹²⁵I-ET-1 and ¹²⁵l-ET-3 binding was totally abolished. However, in the presence of 1 μ M BQ123, which is selective for ET_Areceptors, both bindings were not affected. These results suggest that the human cerebral cortex contains only ET_Breceptors. Cross-linking of ¹²⁵I-ET-1 and ¹²⁵l-ET-3 to membranes with disuccinimidyl suberate resulted in the labeling of two bands of 48 and 31 kDa. Concentrations of IR-ET-1 in the gray and white matter were 7.0 ± 3.2 and 2.5 ± 1.7 fmol/g wet weight, respectively. The demonstration of high-affinity ET_B receptors and the presence of IRET-1 suggest that the peptide may act as a neurotransmitter or neuromodulator in the human cerebral cortex.     

Organellar DNA restriction fragment length polymorphism (RFLP) and nuclear random amplified polymorphic DNA (RAPD) analyses of morphotypes of Gracilaria (Gracilariales,Rhodophyta) from Chile

The extreme phenotypic variability recognized among the species of Gracilaria has highlighted the need for the application of refined methods to help solve taxa identifications. In Chile, there still exists uncertainty about the exact number of Gracilaria species. Our investigations are centered on DNA analyses of morphotypes collected from different geographical locations, namely Lenga and Isla Santa María, Region VIII (36°00 S to 38°00 S), and Maullín, Region X (39°30 S to 43°40 S). These two regions of Chile are considered as areas of confluence of G. chilensis, G. verrucosa, and a species of Gracilariopsis. In this study four morphotypes, from a natural bed located in Maullín, were analyzed for RFLP of plastid DNA and the results compared with data of four morphotypes from a bed in Lenga. The DNA banding patterns from each enzyme digest were identical irrespective of morphotypes and/or locations. In an attempt to unravel the nature of the morphological differences found among Lenga and Maullín morphotypes, RAPD analyses of nuclear DNA were also performed; however, no polymorphism has been found yet. Therefore, the data of this study, as well as concurrent data from preliminary interfertility tests, suggest that all morphotypes belong to a single taxon, Gracilaria chilensis.Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas