The cardiac electrogenic sodium/bicarbonate cotransporter (NBCe1) is activated by aldosterone through the G protein-coupled receptor 30 (GPR 30)

The sodium/bicarbonate cotransporter (NBC) transports extracellular Na⁺ and HCO₃^? into the cytoplasm upon intracellular acidosis, restoring the acidic pH_i to near neutral values. Two different NBC isoforms have been described in the heart, the electroneutral NBCn1 (1Na⁺:1HCO₃^?) and the electrogenic NBCe1 (1Na⁺:2HCO₃^?). Certain non-genomic effects of aldosterone (Ald) were due to an orphan G protein-couple receptor 30 (GPR30). We have recently demonstrated that Ald activates GPR30 in adult rat ventricular myocytes, which transactivates the epidermal growth factor receptor (EGFR) and in turn triggers a reactive oxygen species (ROS)- and PI3K/AKT-dependent pathway, leading to the stimulation of NBC. The aim of this study was to investigate the NBC isoform involved in the Ald/GPR30-induced NBC activation. Using specific NBCe1 inhibitory antibodies (a-L3) we demonstrated that Ald does not affect NBCn1 activity. Ald was able to increase NBCe1 activity recorded in isolation. Using immunofluorescence and confocal microscopy analysis we showed in this work that both NBCe1 and GPR30 are localized in t-tubules. In conclusion, we have demonstrated that NBCe1 is the NBC isoform activated by Ald in the heart.     

Overrepresentation of interactions between homologous proteins in interactomes

It is well proved that the probability that a protein interacts with itself is higher than that it interacts with another protein. It has been recently shown that the probability of interaction is also higher for proteins with significant sequence similarity. In this paper we show that proteins sharing identical PFAM domains interact more often than expected by chance in Saccharomyces cerevisiae and Escherichia coli. We also analyze the variety of domain interfaces used by homologous proteins to interact and show that the overrepresentation of interactions between homological proteins is not caused by small number of pairs of identical "sticky domains" shared between interacting proteins.     

Locally enhanced sampling molecular dynamics study of the dioxygen transport in human cytoglobin

Cytoglobin (Cyg)--a new member of the vertebrate heme globin family--is expressed in many tissues of the human body but its physiological role is still unclear. It may deliver oxygen under hypoxia, serve as a scavenger of reactive species or be involved in collagen synthesis. This protein is usually six-coordinated and binds oxygen by a displacement of the distal HisE7 imidazole. In this paper, the results of 60 ns molecular dynamics (MD) simulations of dioxygen diffusion inside Cyg matrix are discussed. In addition to a classical MD trajectory, an approximate Locally Enhanced Sampling (LES) method has been employed. Classical diffusion paths were carefully analyzed, five cavities in dynamical structures were determined and at least four distinct ligand exit paths were identified. The most probable exit/entry path is connected with a large tunnel present in Cyg. Several residues that are perhaps critical for kinetics of small gaseous diffusion were discovered. A comparison of gaseous ligand transport in Cyg and in the most studied heme protein myoglobin is presented. Implications of efficient oxygen transport found in Cyg to its possible physiological role are discussed.     

Predictors of mortality of patients with acute respiratory failure secondary to chronic obstructive pulmonary disease admitted to an intensive care unit: A one year study

Background

Patients with acute exacerbation of chronic obstructive pulmonary disease (COPD) commonly require hospitalization and admission to intensive care unit (ICU). It is useful to identify patients at the time of admission who are likely to have poor outcome. This study was carried out to define the predictors of mortality in patients with acute exacerbation of COPD and to device a scoring system using the baseline physiological variables for prognosticating these patients.

Methods

Eighty-two patients with acute respiratory failure secondary to COPD admitted to medical ICU over a one-year period were included. Clinical and demographic profile at the time of admission to ICU including APACHE II score and Glasgow coma scale were recorded at the time of admission to ICU. In addition, acid base disorders, renal functions, liver functions and serum albumin, were recorded at the time of presentation. Primary outcome measure was hospital mortality.

Results

Invasive ventilation was required in 69 patients (84.1%). Fifty-two patients survived to hospital discharge (63.4%). APACHE II score at the time of admission to ICU {odds ratio (95 % CI): 1.32 (1.138–1.532); p < 0.001} and serum albumin (done within 24 hours of admission) {odds ratio (95 % CI): 0.114 (0.03-0.432); p = 0.001}. An equation, constructed using the adjusted odds ratio for the two parameters, had an area under the ROC curve of 91.3%. For the choice of cut-off, sensitivity, specificity, positive and negative predictive value for predicting outcome was 90%, 86.5%, 79.4% and 93.7%.

Conclusion

APACHE II score at admission and SA levels with in 24 hrs after admission are independent predictors of mortality for patients with COPD admitted to ICU. The equation derived from these two parameters is useful for predicting outcome of these patients.     

Markers of small cell lung cancer

Lung cancer is the number one cause of cancer death; however, no specific serum biomarker is available till date for detection of early lung cancer. Despite good initial response to chemotherapy, small-cell lung cancer (SCLC) has a poor prognosis. Therefore, it is important to identify molecular markers that might influence survival and may serve as potential therapeutic targets. The review aims to summarize the current knowledge of serum biomarkers in SCLC to improve diagnostic efficiency in the detection of tumor progression in lung cancer. The current knowledge on the known serum cytokines and tumor biomarkers of SCLC is emphasized. Recent findings in the search for novel diagnostic and therapeutic molecular markers using the emerging genomic technology for detecting lung cancer are also described. It is believed that implementing these new research techniques will facilitate and improve early detection, prognostication and better treatment of SCLC.     

Clathrin-mediated endocytosis and recycling of the neuron-specific Na+/H+ exchanger NHE5 isoform. Regulation by phosphatidylinositol 3'-kinase and the actin cytoskeleton

Mammalian Na+/H+ exchangers (NHEs) are a family of integral membrane proteins that play central roles in sodium, acid-base, and cell volume homeostasis. The recently cloned NHE5 isoform is expressed predominantly in brain, but its functional and cellular properties are poorly understood. To facilitate its characterization, an epitope-tagged construct of NHE5 was ectopically expressed in nonneuronal and neuronal cells. In NHE-deficient Chinese hamster ovary AP-1 cells, NHE5 localized at the plasmalemma, but a significant fraction accumulated intracellularly in vesicles that concentrated in a juxtanuclear region. Similarly, in nerve growth factor-differentiated neuroendocrine PC12 cells and primary hippocampal neurons, immunolabeling of NHE5 was detected in endomembrane vesicles in the perinuclear region of the cell body but also along the processes. More detailed characterization in AP-1 cells using organelle-specific markers showed that NHE5 co-localized with internalized transferrin, a marker of recycling endosomes. Transient transfection of a dominant negative mutant of dynamin-1, which inhibits clathrin-mediated endocytosis, blocked uptake of transferrin as well as internalization of NHE5. Likewise, wortmannin inhibition of phosphatidylinositol 3'-kinase, a lipid kinase implicated in endosomal traffic, induced coalescence of vesicles containing NHE5 and caused a pronounced inhibition of plasmalemmal Na+/H+ exchange. By contrast, disruption of the F-actin cytoskeleton with cytochalasin D increased cell surface NHE5 activity and abundance. These observations demonstrate that NHE5 is localized to the recycling endosomal pathway and is dynamically regulated by phosphatidylinositol 3'-kinase and by the state of F-actin assembly.     

Regulation of NHE3 activity by G protein subunits in renal brush-border membranes

NHE3 activity is regulated by phosphorylation/dephosphorylation processes and membrane recycling in intact cells. However, the Na(+)/H(+) exchanger (NHE) can also be regulated by G proteins independent of cytoplasmic second messengers, but the G protein subunits involved in this regulation are not known. Therefore, we studied G protein subunit regulation of NHE3 activity in renal brush-border membrane vesicles (BBMV) in a system devoid of cytoplasmic components and second messengers. Basal NHE3 activity was not regulated by G(s)alpha or G(i)alpha, because antibodies to these G proteins by themselves were without effect. The inhibitory effect of D(1)-like agonists on NHE3 activity was mediated, in part, by G(s)alpha, because it was partially reversed by anti-G(s)alpha antibodies. Moreover, the amount of G(s)alpha that coimmunoprecipitated with NHE3 was increased by fenoldopam in both brush-border membranes and renal proximal tubule cells. Furthermore, guanosine 5'-O-(3-thiotriphosphate) but not guanosine 5'-O-(2-thiodiphosphate), the inactive analog of GDP, increased the amount of G(s)alpha that coimmunoprecipitated with NHE3. The alpha(2)-adrenergic agonist, UK-14304 or pertussis toxin (PTX) alone had no effect on NHE3 activity, but UK-14304 and PTX treatment attenuated the D(1)-like receptor-mediated NHE3 inhibition. The ability of UK-14304 to attenuate the D(1)-like agonist effect was not due to G(i)alpha, because the attenuation was not blocked by anti-G(i)alpha antibodies or by PTX. Anti-Gbeta(common) antibodies, by themselves, slightly inhibited NHE3 activity but had little effect on D(1)-like receptor-mediated NHE3 inhibition. However, anti-Gbeta(common) antibodies reversed the effects of UK-14304 and PTX on D(1)-like agonist-mediated NHE3 inhibition. These studies provide concrete evidence of a direct regulatory role for G(s)alpha, independent of second messengers, in the D(1)-like-mediated inhibition of NHE3 activity in rat renal BBMV. In addition, beta/gamma dimers of heterotrimeric G proteins appear to have a stimulatory effect on NHE3 activity in BBMV.     

Intracellular pH regulation by Na(+)/H(+) exchange requires phosphatidylinositol 4,5-bisphosphate

The carrier-mediated, electroneutral exchange of Na(+) for H(+) across the plasma membrane does not directly consume metabolic energy. Nevertheless, acute depletion of cellular ATP markedly decreases transport. We analyzed the possible involvement of polyphosphoinositides in the metabolic regulation of NHE1, the ubiquitous isoform of the Na(+)/H(+) exchanger. Depletion of ATP was accompanied by a marked reduction of plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP(2)) content. Moreover, sequestration or hydrolysis of plasmalemmal PIP(2), in the absence of ATP depletion, was associated with profound inhibition of NHE1 activity. Examination of the primary structure of the COOH-terminal domain of NHE1 revealed two potential PIP(2)-binding motifs. Fusion proteins encoding these motifs bound PIP(2) in vitro. When transfected into antiport-deficient cells, mutant forms of NHE1 lacking the putative PIP(2)-binding domains had greatly reduced transport capability, implying that association with PIP(2) is required for optimal activity. These findings suggest that NHE1 activity is modulated by phosphoinositides and that the inhibitory effect of ATP depletion may be attributable, at least in part, to the accompanying net dephosphorylation of PIP(2).     

Catalytic activities of the 20 S proteasome, a multicatalytic proteinase complex

The proteasome, a multisubunit, multicatalytic proteinase complex, is attracting growing attention as the main intracellular, extralysosomal, proteolytic system involved in ubiquitin-(Ub) dependent and Ub-independent intracellular proteolysis. Its involvement in the mitotic cycle, and control of the half-life of most cellular proteins, functions absolutely necessary for cell growth and viability, make it an attractive target for researchers of intracellular metabolism and an important target for pharmacological intervention. The proteasome belongs to a new mechanistic class of proteases, the N-terminal nucleophile hydrolases, where the N-terminal threonine residue functions as the nucleophile. This minireview focuses on the three classical catalytic activities of the proteasome, designated chymotrypsin-like, trypsin-like, and peptidyl-glutamyl-peptide hydrolyzing in eukaryotes and also the activities of the more simple Archaebacteria and Eubacteria proteasomes. Other catalytic activities of the proteasome and their possible origin are also examined. The specificity of the catalytic components toward synthetic substrates, natural peptides, and proteins and their relationship to the catalytic centers are reviewed. Some unanswered questions and future research directions are suggested.