MiR-101 and miR-144 Regulate the Expression of the CFTR Chloride Channel in the Lung

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that plays a critical role in the lung by maintaining fluid homeostasis. Absence or malfunction of CFTR leads to Cystic Fibrosis, a disease characterized by chronic infection and inflammation. We recently reported that air pollutants such as cigarette smoke and cadmium negatively regulate the expression of CFTR by affecting several steps in the biogenesis of CFTR protein. MicroRNAs (miRNAs) have recently received a great deal of attention as both biomarkers and therapeutics due to their ability to regulate multiple genes. Here, we show that cigarette smoke and cadmium up-regulate the expression of two miRNAs (miR-101 and miR-144) that are predicted to target CFTR in human bronchial epithelial cells. When premature miR-101 and miR-144 were transfected in human airway epithelial cells, they directly targeted the CFTR 3′UTR and suppressed the expression of the CFTR protein. Since miR-101 was highly up-regulated by cigarette smoke in vitro, we investigated whether such increase also occurred in vivo. Mice exposed to cigarette smoke for 4 weeks demonstrated an up-regulation of miR-101 and suppression of CFTR protein in their lungs. Finally, we show that miR-101 is highly expressed in lung samples from patients with severe chronic obstructive pulmonary disease (COPD) when compared to control patients. Taken together, these results suggest that chronic cigarette smoking up-regulates miR-101 and that this miRNA could contribute to suppression of CFTR in the lungs of COPD patients.     

Identification of Amino Acid Residues in the α, β, and γ Subunits of the Epithelial Sodium Channel (ENaC) Involved in Amiloride Block and Ion Permeation

The amiloride-sensitive epithelial Nachannel (ENaC) is a heteromultimeric channel made of three αβγ subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in β and γ subunits at position βG525 and γG537 increased the apparent inhibitory constant (K _i) for amiloride by >1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the α subunit increased amiloride K _i by 20-fold, without changing channel conducting properties. Coexpression of these mutated αβγ subunits resulted in a nonconducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by externalZn²⁺ ions, in particular the αS583C mutant showing a K _i for Zn²⁺of 29 μM. Mutations of residues αW582L or βG522D also increased amiloride K _i, the later mutation generating a Ca²⁺blocking site located 15% within the membrane electric field. These experiments provide strong evidence that αβγ ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of αβγ subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na⁺ions at an external Na⁺binding site preventing ion permeation through the channel pore.     

Differential signaling by adaptor molecules LRP1 and ShcA regulates adipogenesis by the insulin-like growth factor-1 receptor

The low density lipoprotein receptor-related protein (LRP1) is a transmembrane receptor that integrates multiple signaling pathways. Its cytoplasmic domain serves as docking sites for several adaptor proteins such as the Src homology 2/α-collagen (ShcA), which also binds to several tyrosine kinase receptors such as the insulin-like growth factor 1 (IGF-1) receptor. However, the physiological significance of the physical interaction between LRP1 and ShcA, and whether this interaction modifies tyrosine kinase receptor signaling, are still unknown. Here we report that LRP1 forms a complex with the IGF-1 receptor, and that LRP1 is required for ShcA to become sensitive to IGF-1 stimulation. Upon IGF-1 treatment, ShcA is tyrosine phosphorylated and translocates to the plasma membrane only in the presence of LRP1. This leads to the recruitment of the growth factor receptor-bound protein 2 (Grb2) to ShcA, and activation of the Ras/MAP kinase pathway. Conversely, in the absence of ShcA, IGF-1 signaling bifurcates toward the Akt/mammalian target of rapamycin pathway and accelerates adipocyte differentiation when cells are stimulated for adipogenesis. These results establish the LRP1-ShcA complex as an essential component in the IGF-1-regulated pathway for MAP kinase and Akt/mammalian target of rapamycin activation, and may help to understand the IGF-1 signaling shift from clonal expansion to growth-arrested cells and differentiation during adipogenesis.     

A new seabird (Aves, cf. Phaethontidae) from the Lower Eocene phosphates of Morocco

A proximal part of humerus from the basal Ypresian (lowermost Eocene) of the Ouled Abdoun Basin, Morocco, is described as a new genus and species tentatively assigned to the Phaethontidae (tropicbirds). This fossil possibly represents the oldest record of the Phaethontidae and markedly differs from Lithoptila, a contemporaneous Prophaethontidae from the same locality. This new taxon lived in a tropical climate and was probably an efficient flier with pelagic habits, like extant tropicbirds of the genus Phaethon.     

Auxin and ethylene promote root hair elongation in Arabidopsis

Genetic and physiological studies implicate the phytohormones auxin and ethylene in root hair development. To learn more about the role of these compounds, we have examined the root hair phenotype of a number of auxin- and ethylene-related mutants. In a previous study, Masucci and Schiefelbein (1996) showed that neither the auxin response mutations aux1 and axr1 nor the ethylene response mutations etr1 and ein2 have a significant effect on root hair initiation. In this study, we found that mutants deficient in either auxin or ethylene response have a pronounced effect on root hair length. Treatment of wild-type, axr1 and etr1 seedlings with the synthetic auxin, 2,4-D, or the ethylene precursor ACC, led to the development of longer root hairs than untreated seedlings. Furthermore, axr1 seedlings grown in the presence of ACC produce ectopic root hairs and an unusual pattern of long root hairs followed by regions that completely lack root hairs. These studies indicate that both auxin and ethylene are required for normal root hair elongation.     

Contribution of leaf surface fungi to the air spora

High concentrations of airborne fungal spores frequently occur from spring through fall in temperate areas of the world. Although it is generally assumed that fungi on leaf surfaces are contributors to the air spora, little data are available comparing the types of fungi found on leaf surfaces with those in the atmosphere. Air sampling was carried out with a Burkard Spore Trap located on the roof of a building on the University of Tulsa campus using standard methods. Leaf samples were aseptically collected from Ulmus americana and Quercus palustris trees on campus, placed in sterile plastic bags, and brought to the lab. For each leaf, 4 cm² areas of both upper and lower leaf surfaces were swabbed and plated on malt extract agar with streptomycin. Cultures were incubated at room temperature for 5–7 days and then examined microscopically. Results were expressed as colony forming units (CFU)/cm². Twenty-one fungal taxa were identified from the air samples. The most abundant taxa were Cladosporium, ascospores, basidiospores, and Alternaria; together these four spore types comprised over 90% of the yearly total. Yeasts were the most abundant fungi isolated from both leaf types. Among the mycelial fungi were Phoma species, followed by Cladosporium and Alternaria. Overall twenty genera of filamentous fungi were identified. Yeasts and Phoma are normally splash dispersed and were not identified in the Burkard air samples. However, 10 taxa isolated from leaf surfaces were registered in air samples. Crude estimates of the leaf surface area of each tree suggest that the total fungal load was approximately 5.04×10⁸ CFU for Ulmus and 2.71×10⁸ CFU for Quercus. Of these levels, 19% were from fungi also detected in air samples. The data suggest that some leaf-surface fungi are major contributors to the air spora.     

The air spora close to a compost facility in Northeast Oklahoma: Part I—spore trap sampling

A compost facility in northeast Oklahoma is located relatively close to a residential area and is the focus of complaints about smell and concerns about health effects. Several species of Aspergillus have been known to cause health problems, and at least one of these species is dominant in compost. The atmosphere surrounding the compost facility was monitored for 1 year using Burkard spore traps to determine if there was a significant difference in Penicillium/Aspergillus type spores concentration between a test and control site. Samplers were situated 710 m downwind for the test site and 6,085 m upwind at the control site. There was no significant difference in mean concentration of Penicillium/Aspergillus type spores between the two sites (t = 0.576 P > 0.05). The mean concentration of total spores was significantly higher at the upwind control site (t = −7.64, P < 0.01). Wind direction was examined to determine if the compost facility was a possible source for any spikes in concentration. No clear relationship was found between wind direction and mean Penicillium/Aspergillus concentration at the test site, but peak concentrations of Penicillium/Aspergillus seen at the test site were on days when it was downwind from the composting facility. However, these concentrations were no higher than those seen at the control site on other days. If the compost was releasing large amounts of Penicillium/Aspergillus type spores into the atmosphere they were generally diluted to background levels by the time they reached the test site.