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Research has been conducted on adenovirus receptors, CAR. Results demonstrate that adenovirus uses CAR only for the entry into host cells and for escape across epithelial barriers to the environment.

Impaired bactericidal activity of airway surface fluid in cystic fibrosis (CF) patients is caused by defective Cl transport across airway epithelia. Bactericidal activity in normal airway surface fluid depends on a low salt concentration. In CF epithelia, however, loss of the phosphorylation-regulated Cl channels results in an abnormally high salt concentration in the airway surface fluid, which reduces bactericidal activity.

Liddle's syndrome is due to mutations in the C-terminal of the human epithelial Na+ channel (hENaC) subunits which causes hypertension by increasing Na+ absorption. The increased Na+ absorption is due to an increase in the number of Na+ channels in the epithelium. The conductance of individual channels is unaltered. The mutation is the deletion of conserved sequences in the C-terminal which control the internalization of hENaC.

Mutant D836X that encodes the amino-terminal region of the cystic fibrosis transmembrane conductance regulator (CFTR) with a membrane-spanning domain and a nucleotide binding domain NBD. The R Domain constitutes a controlled chloride channel with characteristics that are similar to that of the wild-type channel. Activity increased with phosphorylation but conductance did not require phosphorylation.

Therapeutic treatment administered to three patients with cystic fibrosis (CF) involved the incorporation of an E1-defective adenovirus into the nasal airway epithelium. CF, attributed to mutations in the gene coding for CF transmembrane conductance regulator, was characterized by a dysfunction in the chloride transport mechanism. The administration of adenovirus-regulated gene transfer therapy was effective in treating the malfunctioning of Cl-transport channels. The treatment resulted in a decline in the high basal transepithelial voltage and stabilized its sensitivity to cyclic adenosine monophosphate.

The four processes that allow mutations to interfere with the cystic fibrosis transmembrane conductance regulator (CFTR) are faulty control of CFTR, erroneous conduction, faulty protein processing and faulty protein generation. The major differences in clinical phenotype and the problems involved in forecasting clinical irregularities caused by CF-associated mutations are some of the reasons for the difficulty of categorizing CFTR mutations using biochemical dysfunction as the criterion.

Cystic fibrosis is caused by cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations, the most common of which is the deletion of phenylalanine at position 508 (deltaF508). STE6-CFTR hybrid genes in yeast cells were used to identify revertants of CFTRdeltaF508 mutations. When deltaF508 was introduced into the first nucleotide-binding domain (NBD1) of the yeast STE6 a-factor transporter, mating efficiency decreased. Two deltaF508 revertant mutations within the CFTR NBD1 sequence restored mating and introduction of these revertants into human CFTR partially restored the processing and Cl- channel gating defects.

Ca(super 2+) toxicity remains the central focus of ischemic brain injury, and acidosis is a common feature of ischemia. The study shows that acidosis activates Ca(super 2+)-permeable acid-sensing ion channels (ASICs).

Research demonstrates that the regulation of channel activity of the cystic fibrosis transmemebrane conductance regulator (CFTR), an anion channel protein of the ABC transporter family, is regulated by ATPase and Adenylate kinase. However, at physiologic nucleotide concentrations, the latter enzyme controls gating.

Investigates the adenylate kinase activity in adenosine triphosphate-binding cassette (ABC) transporters. Description and characteristics of ABC transporters; Implications of adenylate kinase activity for nucleotide-binding domain dimerization; Mechanisms that enable ABC transporters to alter adenosine triphosphate, adenosine diphosphate or adenylic acid concentrations.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane-spanning adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter. ABC transporters and other nuclear and cytoplasmic ABC proteins have ATPase activity that is coupled to their biological function. Recent studies with CFTR and two nonmembrane-bound ABC proteins, the DNA repair enzyme Rad50 and a structural maintenance of chromosome (SMC) protein, challenge the model that the function of all ABC proteins depends solely on their associated ATPase activity. Patch clamp studies indicated that in the presence of physiologically relevant concentrations of adenosine 5'-monophosphate (AMP), CFTR Cl_ channel function is coupled to adenylate kinase activity (ATP+AMP⇆2 ADP). Work with Rad50 and SMC showed that these enzymes catalyze both ATPase and adenylate kinase reactions. However, despite the supportive electrophysiological results with CFTR, there are no biochemical data demonstrating intrinsic adenylate kinase activity of a membrane-bound ABC transporter. We developed a biochemical assay for adenylate kinase activity, in which the radioactive γ-phosphate of a nucleotide triphosphate could transfer to a photo activatable AMP analog. UV irradiation could then trap the 32P on the adenylate kinase. With this assay, we discovered phosphoryl group transfer that labeled CFTR, thereby demonstrating its adenylate kinase activity. Our results also suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for adenylate kinase activity. These biochemical data complement earlier biophysical studies of CFTR and indicate that the ABC transporter CFTR can function as an adenylate kinase. [ABSTRACT FROM AUTHOR]

Extracellular acid can have important effects on neuron function. In central and peripheral neurons, acid-sensing ion channels (ASICs) have emerged as key receptors for extracellular protons, and recent studies suggest diverse roles for these channels in the pathophysiology of pain, ischemic stroke and psychiatric disease. ASICs have also been implicated in mechanosensation in the peripheral nervous system and in neurotransmission in the central nervous system. Here, we briefly review advances in our understanding of ASICs, their potential contributions to disease, and the possibility for their therapeutic modification. [Copyright &y& Elsevier]

Two enzymes, protein phosphatase 2A and atypical protein kinase C, are associated with the tight junction and regulate its function. For example, phosphorylation of the tight junction protein occludin is required for its incorporation into the junction. The association of a kinase and phosphatase with the tight junction suggests that a balance between their activities exists and is required for normal tight junction function. This hypothesis predicts that loss of epithelial integrity may disrupt this balance in such a way as to facilitate restoration of epithelial integrity. Our previous data have shown that apically localized growth factors segregate from their basolaterally localized erbB receptors. Loss of epithelial integrity allows ligand access to the basolateral membrane where it immediately binds to and activates erbB receptors. We found that activation of erbB1 leads to phosphorylation of protein phosphatase 2A, inhibiting its activity. Importantly, this phosphorylation event was dependent on factors in the overlying airway surface liquid; washing away this liquid prevented phosphorylation, erbB1-mediated inhibition of phosphatase activity would shift the balance in favor of the kinase such that tight junction proteins would regain their phosphorylation, allowing for their incorporation into the junction complex. This mechanism provides a rapid means of sensing the loss of epithelial integrity and subsequently restoring barrier function. [ABSTRACT FROM AUTHOR]

Background: Impaired brain energy metabolism is a key feature of Parkinson's disease (PD). Terazosin (TZ) binds phosphoglycerate kinase 1 and stimulates its activity, which enhances glycolysis and increases ATP levels. Preclinical and epidemiologic data suggest that TZ may be neuroprotective in PD. We aimed to assess target engagement and safety of TZ in people with PD.Methods: We performed a 12-week pilot study in people with PD. Participants were randomized to receive 5 mg TZ or placebo. Participants and study personnel were blinded. We assessed TZ target engagement by measuring brain ATP with 31P-magnetic resonance spectroscopy (MRS) and whole blood ATP with a luminescence assay. Robust linear regression models compared changes between groups controlling for baseline brain and blood ATP levels, respectively. We also assessed clinical measures of PD and adverse events.Results: Thirteen participants were randomized. Mild dizziness/lightheadedness was more common in the TZ group, and three participants taking TZ dropped out because of dizziness and/or orthostatic hypotension. Compared to the placebo group, the TZ group had a significant increase in the ratio of βATP to inorganic phosphate in the brain. The TZ group also had a significant increase in blood ATP levels compared to the placebo group (p < 0.01).Conclusions: This pilot study suggests that TZ may engage its target and change ATP levels in the brain and blood of people with PD. Further studies may be warranted to test the disease-modifying potential of TZ. [ABSTRACT FROM AUTHOR]

Pulmonary neuroendocrine cells (PNECs) are rare airway cells with potential sensory capacity linked to vagal neurons and immune cells. How PNECs sense and respond to external stimuli remains poorly understood. We discovered PNECs located within pig and human submucosal glands, a tissue that produces much of the mucus that defends the lung. These PNECs sense succinate, an inflammatory molecule in liquid lining the airway surface. The results indicate that succinate migrates down the submucosal gland duct to the acinus, where it triggers apical succinate receptors, causing PNECs to release ATP. The short-range ATP signal stimulates the contraction of myoepithelial cells wrapped tightly around the submucosal glands. Succinate-triggered gland contraction may complement the action of neurotransmitters that induce mucus release but not gland contraction to promote mucus ejection onto the airway surface. These findings identify a local circuit in which rare PNECs within submucosal glands sense an environmental cue to orchestrate the function of airway glands. [Display omitted] • PNECs localize in airway submucosal glands of humans and pigs • Submucosal PNECs are chemosensory cells that express SUCNR1 • PNECs release ATP in response to luminal succinate • Extracellular ATP from PNECs triggers myoepithelial cell contraction via P2Y1R Yu et al. find that, in addition to the airway surface, pulmonary neuroendocrine cells (PNECs) localize in the submucosal glands of pigs and humans. PNECs express SUCNR1 to sense luminal succinate and trigger ATP release. ATP stimulates myoepithelial cell contraction via P2Y1 receptors, resulting in glandular mucus ejection. [ABSTRACT FROM AUTHOR]

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P°,P5-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl- channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. [ABSTRACT FROM AUTHOR]

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases,we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5'-triphosphate (8-N3-ATP) and 8-azidoadenosine 5'-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced pho- to labeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P¹,P5 -di(adenosine-5) pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2. [ABSTRACT FROM AUTHOR]

Operons are primarily a bacterial phenomenon, not commonly observed in eukaryotes. However, new research indicates that operons are found in higher organisms as well. There are instances of operons found in C. elegans, Drosophila melanogaster and other eukaryotic species. We developed a prototype using positional, structural and gene expression information to identify candidate operons. We focused our efforts on "trans-spliced" operons in which the pre-mRNA is trans-spliced into individual transcripts and subsequently translated, as widely observed in C. elegans and some instances in Drosophila. We identify several candidate operons in Drosophila melanogaster of which two have been subsequently molecularly validated. [ABSTRACT FROM AUTHOR]

Abstract: A recently discovered enzyme system produces antibacterial hypothiocyanite (OSCN−) in the airway lumen by oxidizing the secreted precursor thiocyanate (SCN−). Airway epithelial cultures have been shown to secrete SCN− in a CFTR-dependent manner. Thus, reduced SCN− availability in the airway might contribute to the pathogenesis of cystic fibrosis (CF), a disease caused by mutations in the CFTR gene and characterized by an airway host defense defect. We tested this hypothesis by analyzing the SCN− concentration in the nasal airway surface liquid (ASL) of CF patients and non-CF subjects and in the tracheobronchial ASL of CFTR-ΔF508 homozygous pigs and control littermates. In the nasal ASL, the SCN− concentration was ~30-fold higher than in serum independent of the CFTR mutation status of the human subject. In the tracheobronchial ASL of CF pigs, the SCN− concentration was somewhat reduced. Among human subjects, SCN− concentrations in the ASL varied from person to person independent of CFTR expression, and CF patients with high SCN− levels had better lung function than those with low SCN− levels. Thus, although CFTR can contribute to SCN− transport, it is not indispensable for the high SCN− concentration in ASL. The correlation between lung function and SCN− concentration in CF patients may reflect a beneficial role for SCN−. [Copyright &y& Elsevier]

The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and peripheral neurons where it generates transient cation currents when extracellular pH falls. ASIC1a confers pHdependent modulation on postsynaptic dendritic spines and has critical effects in neurological diseases associated with a reduced pH. However, knowledge of the proteins that interact with ASIC1a and influence its function is limited. Here, we show that α-actinin, which links membrane proteins to the actin cytoskeleton, associates with ASIC1a in brain and in cultured cells. The interaction depended on an α-actinin-binding site in the ASIC1a C terminus that was specific for ASIC1a versus other ASICs and for α-actinin-1 and -4. Co-expressing α-actinin-4 altered ASIC1a current density, pH sensitivity, desensitization rate, and recovery from desensitization. Moreover, reducing α-actinin expression altered acid-activated currents in hippocampal neurons. These findings suggest that α-actinins may link ASIC1a to a macromolecular complex in the postsynaptic membrane where it regulates ASIC1a activity. [ABSTRACT FROM AUTHOR]

Three mutations (R120G, Q151X, and 464delCT) in the small heat shock protein αβ-crystallin cause inherited myofibrillar myopathy. In an effort to elucidate the molecular basis for the associated myopathy, we have determined the following for these mutant αβ-crystallin proteins: (i) the formation of aggregates in transfected cells; (ii) the partition into different subcellular fractions; (iii) the phosphorylation status; and (iv) the ability to interact with themselves, with wild-type αβ-crystaffin, and with other small heat shock proteins that are abundant in muscles. We found that all three αβ-crystallin mutants have an increased tendency to form cytoplasmic aggregates in transfected cells and significantly increased levels of phosphorylation when compared with the wild-type protein. Although wild-type αβ-crystallin partitioned essentially into the cytosol and membranes/organelles fractions, mutant αβ-crystallin proteins partitioned additionally into the nuclear and cytoskeletal fractions. By using various protein interaction assays, including quantitative fluorescence resonance energy transfer measurements in live cells, we found abnormal interactions of the various αβ-crystallin mutants with wild-type αβ-crystallin, with themselves, and with the other small heat shock proteins Hsp20, Hsp22, and possibly with Hsp27. The collected data suggest that each αβ-crystallin mutant has a unique pattern of abnormal interaction properties. These distinct properties of the αβ-crystallin mutants identified are likely to contribute to a better understanding of the gradual manifestation and clinical heterogeneity of the associated myopathy in patients. [ABSTRACT FROM AUTHOR]

The acid-sensing ion channel-3 (ASIC3) is a degenerin/ epithelial sodium channel expressed in the peripheral nervous system. Previous studies indicate that it participates in the response to mechanical and painful stimuli, perhaps contributing to mechanoreceptor and/or H+gated nociceptor function. ASIC3 subunits contain intracellular N and C termini that may control channel localization and function. We found that a PDZ-binding motif at the ASIC3 C terminus interacts with four different proteins that contain PDZ domains: PSD-95, Lin7b, MAGI-1b, and PIST. ASIC3 and these interacting proreins were expressed in dorsal root ganglia and spinal cord, and PSD-95 co-precipitated ASIC3 from spinal cord. When expressed in heterologous cells, PSD-95 reduced the amplitude of ASIC3 acid-evoked currents, whereas Lin-7b increased current amplitude. PSD-95 and Lin-7b altered current density by decreasing or increasing, respectively, the amount of ASIC3 on the cell surface. The finding that multiple PDZ-containing proteins bind ASIC3 and can influence its presence in the plasma membrane suggests that they may play an important role in the contribution of ASIC3 to nociception and mechanosensation. [ABSTRACT FROM AUTHOR]

In rodent whole embryo culture (WEC), finding vehicles for non-aqueous-soluble compounds has been problematic due to developmental toxicity associated with many solvents. The purpose of this study was to identify alternative vehicles for insoluble compounds. In WEC, we evaluated carrier solutions containing bovine serum albumin (BSA) and glycerol as well as the solvents, formamide, dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and ethanol, for relative teratogenicity and delivery of the insoluble teratogen, all-trans retinoic acid (RA). At a concentration of ≤0.04%, formamide and DMF exhibited no significant toxicity to cultured rat embryos and were effective at delivering RA to the embryo. The BSA and glycerol carrier solutions were not teratogenic, although both inhibited robust formation of yolk sac vasculature. Both solutions delivered RA to the cultured rat embryos at higher doses. In summary, all four solvents/solutions may have utility as vehicles dependent upon the chemical properties of the compound to be solubilized. [Copyright &y& Elsevier]

Hippocampal neurons express subunits of the acid-sensing ion channel (ASIC1 and ASIC2) and exhibit large cation currents that are transiently activated by acidic extracellular solutions. Earlier work indicated that ASIC1 contributed to the current in these neurons and suggested its importance for normal behavior. However, the specific contribution of ASIC1 and ASIC2 subunits to acid-evoked currents in hippocampal neurons remained uncertain. To decipher the individual role of the ASIC subunits, we studied H+-gated currents in neurons from both ASIC1 and ASIC2 null mice. We found that much of the current was produced by ASICla/2a heteromultimeric channels, and individual subunits made distinct contributions. The ASIC1a subunit was key in establishing current amplitude. The ASIC2a subunit had little effect on amplitude but influenced desensitization, recovery from desensitization, pH sensitivity, and the response to modulatory agents. We also found heterogeneity in the contribution of ASIC2 throughout the neuronal population, with individual neurons expressing both ASIC1a homomultimeric and ASIC1a/2a heteromultimeric channels. Studies of neurons heterozygous for disrupted ASIC alleles indicated that the properties of H+-gated currents are dependent on the proportion of the individual subunits. These findings indicate that the absolute and relative amounts of ASIC subunits determine the amplitude and properties of hippocampal H+-gated currents and therefore may contribute to normal physiology and pathophysiology. [ABSTRACT FROM AUTHOR]

Mammalian small heat shock proteins (sHSP) are abundant in muscles and are implicated in both muscle function and myopathies. Recently a new sHSP, HSP22 (HSPB8, H11), was identified in the human heart by its interaction with HSP27 (HSPB1). Using phylogenetic analysis we show that HSP22 is a true member of the sHSP superfamily, sHSPs interact with each other and form homo- and hetero-oligomeric complexes. The function of these complexes is poorly understood. Using gel filtration HPLC, the yeast two-hybrid method, immunoprecipitation, cross-linking, and fluorescence resonance energy transfer microscopy, we report that (i) HSP22 forms high molecular mass complexes in the heart, (ii) HSP22 interacts with itself, cvHSP (HSPB7), MKBP (HSPB2) and HSP27, and (iii) HSP22 has two binding domains (N- and C-terminal) that are specific for different binding partners. HSP22 homo-dimers are formed through N-N and N-C interactions, and HSP22-cvHSP heterodimers through C-C interaction. HSP22-MKBP and HSP22-HSP27 heterodimers involve the N and C termini of HSP22 and HSP27, respectively, but appear to require full-length protein as a binding partner. [ABSTRACT FROM AUTHOR]

In response to respiratory insults, airway submucosal glands secrete copious mucus strands to increase mucociliary clearance and protect the lung. However, in cystic fibrosis, stimulating submucosal glands has the opposite effect, disrupting mucociliary transport. In cystic fibrosis (CF) pigs, loss of cystic fibrosis transmembrane conductance regulator (CFTR) anion channels produced submucosal gland mucus that was abnormally acidic with an increased protein concentration. To test whether these variables alter mucus, we produced a microfluidic model of submucosal glands using mucus vesicles from banana slugs. Acidic pH and increased protein concentration decreased mucus gel volume and increased mucus strand elasticity and tensile strength. However, once mucus strands were formed, changing pH or protein concentration largely failed to alter the biophysical properties. Likewise, raising pH or apical perfusion did not improve clearance of mucus strands from CF airways. These findings reveal mechanisms responsible for impaired mucociliary transport in CF and have important implications for potential treatments. • Cystic fibrosis submucosal glands produce abnormally acidic and concentrated mucus • A microfluidic model using banana slug mucus produced strands of mucus • Acidic, concentrated mucus formed strands with high elasticity and tensile strength • After mucus strands formed, their biophysical properties resisted further change In cystic fibrosis, airway submucosal glands produce mucus that disrupts mucociliary transport. Using pigs, which have lungs like humans, and a microfluidic model of glands, Xie et al. show that acidic pH and increased protein concentration increase the elasticity and tensile strength of mucus strands, thereby preventing normal mucociliary transport. [ABSTRACT FROM AUTHOR]