Postnatal glucocorticoids induce alpha-ENaC formation and regulate glucocorticoid receptors in the preterm rabbit lung

At birth, lung fluid clearance is coupled to Na+ transport through epithelial Na+ channels (ENaC) in the distal lung epithelium. We evaluated the effect of postnatal glucocorticoids (GC) on lung alpha-ENaC expression in preterm 29-day gestational age (GA) fetal rabbits. Postnatal treatment of 29-day GA fetuses with 0.5 mg/kg of dexamethasone (Dex) iv resulted in a 2- and 22-fold increase in lung alpha-ENaC mRNA expression compared with saline-treated fetuses after 8 and 16 h, respectively. Lung alpha-ENaC protein levels in Dex-treated fetuses were also elevated compared with saline-treated counterparts. The extravascular lung water (EVLW)/dry lung tissue weight ratios of 29-day GA fetuses treated with either saline or Dex decreased over 24 h compared with that observed at birth; however, at 24 h, the EVLW/dry lung tissue weight ratios of saline- and Dex-treated fetuses were similar. Dex-induced alpha-ENaC mRNA and protein levels were attenuated by glucocorticoid receptor (GCR) antagonist RU-486 in fetal distal lung epithelial cells isolated from 29-day GA fetuses, indicating that GC-dependent augmentation of lung alpha-ENaC requires the presence of functional GCR. Lung GCR mRNA expression and protein levels were elevated in 29-day GA fetuses compared with fetuses at earlier GA. Exposure of 29-day GA fetuses to Dex for 16 h caused a 2.1-fold increase in lung GCR mRNA expression, but GCR protein levels were decreased in Dex-treated fetuses after 24 h. We conclude that postnatal treatment of preterm 29-day GA fetal rabbits with GC results in an elevation of lung alpha-ENaC accompanied by an autoregulation of pulmonary GCR.     

The chemokine receptor CXCR3 and its splice variant are expressed in human airway epithelial cells

Activation of the chemokine receptor CXCR3 by its cognate ligands induces several differentiated cellular responses important to the growth and migration of a variety of hematopoietic and structural cells. In the human respiratory tract, human airway epithelial cells (HAEC) release the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. Simultaneous expression of CXCR3 by HAEC would have important implications for the processes of airway inflammation and repair. Accordingly, in the present study we sought to determine whether HAEC also express the classic CXCR3 chemokine receptor CXCR3-A and its splice variant CXCR3-B and hence may respond in autocrine fashion to its ligands. We found that cultured HAEC (16-HBE and tracheocytes) constitutively expressed CXCR3 mRNA and protein. CXCR3 mRNA levels assessed by expression array were approximately 35% of beta-actin expression. In contrast, CCR3, CCR4, CCR5, CCR8, and CX3CR1 were <5% beta-actin. Both CXCR3-A and -B were expressed. Furthermore, tracheocytes freshly harvested by bronchoscopy stained positively for CXCR3 by immunofluorescence microscopy, and 68% of cytokeratin-positive tracheocytes (i.e., the epithelial cell population) were positive for CXCR3 by flow cytometry. In 16-HBE cells, CXCR3 receptor density was approximately 78,000 receptors/cell when assessed by competitive displacement of 125I-labeled IP-10/CXCL10. Finally, CXCR3 ligands induced chemotactic responses and actin reorganization in 16-HBE cells. These findings indicate constitutive expression by HAEC of a functional CXC chemokine receptor, CXCR3. Our data suggest the possibility that autocrine activation of CXCR3 expressed by HAEC may contribute to airway inflammation and remodeling in obstructive lung disease by regulating HAEC migration.     

Smad and p38-MAPK signaling mediates apoptotic effects of transforming growth factor-beta1 in human airway epithelial cells

Transforming growth factor-beta1 (TGF-beta1) belongs to a family of multifunctional cytokines that regulate a variety of biological processes, including cell differentiation, proliferation, and apoptosis. The effects of TGF-beta1 are cell context and cell cycle specific and may be signaled through several pathways. We examined the effect of TGF-beta1 on apoptosis of primary human central airway epithelial cells and cell lines. TGF-beta1 protected human airway epithelial cells from apoptosis induced by either activation of the Fas death receptor (CD95) or by corticosteroids. This protective effect was blocked by inhibition of the Smad pathway via overexpression of inhibitory Smad7. The protective effect is associated with an increase in the cyclin-dependent kinase inhibitor p21 and was blocked by the overexpression of key gatekeeper cyclins for the G1/S interface, cyclins D1 and E. Blockade of the Smad pathway by overexpression of the inhibitory Smad7 permitted demonstration of a TGF-beta-mediated proapoptotic pathway. This proapoptotic effect was blocked by inhibition of the p38 MAPK kinase signaling with the inhibitor SB-203580 and was associated with an increase in p38 activity as measured by a kinase assay. Here we demonstrate dual signaling pathways involving TGF-beta1, an antiapoptotic pathway mediated by the Smad pathway involving p21, and an apoptosis-permissive pathway mediated in part by p38 MAPK.     

High-level expression, purification, and characterization of recombinant wheat xylanase inhibitor TAXI-I secreted by the yeast Pichia pastoris

Triticum aestivum xylanase inhibitor I (TAXI-I) is a wheat protein that inhibits microbial xylanases belonging to glycoside hydrolase family 11. In the present study, recombinant TAXI-I (rTAXI-I) was successfully produced by the methylotrophic yeast Pichia pastoris at high expression levels (approximately 75 mg/L). The rTAXI-I protein was purified from the P. pastoris culture medium using cation exchange and gel filtration chromatographic steps. rTAXI-I has an iso-electric point of at least 9.3 and a mass spectrometry molecular mass of 42,013 Da indicative of one N-linked glycosylation. The recombinant protein fold was confirmed by circular dichroism spectroscopy. Xylanase inhibition by rTAXI-I was optimal at 20-30 degrees C and at pH 5.0. rTAXI-I still showed xylanase inhibition activity at 30 degrees C after a 40 min pre-incubation step at temperatures between 4 and 70 degrees C and after 2 h pre-incubation at room temperature at a pH ranging from 3.0 to 12.0, respectively. All tested glycoside hydrolase family 11 xylanases were inhibited by rTAXI-I whereas those belonging to family 10 were not. Specific inhibition activities against family 11 Aspergillus niger and Bacillus subtilis xylanases were 3570 and 2940IU/mg protein, respectively. The obtained biochemical characteristics of rTAXI-I produced by P. pastoris (no proteolytical cleft) were similar to those of natural TAXI-I (mixture of proteolytically processed and non-processed forms) and non-glycosylated rTAXI-I expressed in Escherichia coli. The present results show that xylanase inhibition activity of TAXI-I is only affected to a limited degree by its glycosylation or proteolytic processing.     

A 9 angstroms single particle reconstruction from CCD captured images on a 200 kV electron cryomicroscope

Sub-nanometer resolution structure determination is becoming a common practice in electron cryomicroscopy of macromolecular assemblies. The data for these studies have until now been collected on photographic film. Using cytoplasmic polyhedrosis virus (CPV), a previously determined structure, as a test specimen, we show the feasibility of obtaining a 9 angstroms structure from images acquired from a 4 k x 4 k Gatan CCD on a 200 kV electron cryomicroscope. The match of the alpha-helices in the protein components of the CPV with the previous structure of the same virus validates the suitability of this type of camera as the recording media targeted for single particle reconstructions at sub-nanometer resolution.     

The N-terminal substrate-binding domain of ClpA unfoldase is highly mobile and extends axially from the distal surface of ClpAP protease

ClpAP is a barrel-like complex consisting of hexameric rings of the ClpA ATPase stacked on the double heptameric ring of ClpP peptidase. ClpA has two AAA+ domains (Dl and D2) and a 153-residue N-domain. Substrate proteins bind to the distal surface of ClpA and are unfolded and translocated axially into ClpP. To gain insight into the functional architecture of ClpA in the ATPgammaS state, we have determined its structure at 12A resolution by cryo-electron microscopy. The resulting model has two tiers, corresponding to rings of Dl and D2 domains: oddly, there is no sign of the N-domains in the density map. However, they were detected as faint diffuse density distal to the Dl tier in a difference image between wild-type ClpAP and a mutant lacking the N-domain. This region is also accentuated in a variance map of ClpAP and in a difference imaging experiment with ClpAP complexed with ClpS, a 12kDa protein that binds to the N-domain. These observations demonstrate that the N-domains are highly mobile. From molecular modeling, we identify their median position and estimate that they undergo fluctuations of at least 30A. We discuss the implications of these observations for the role of N-domains in substrate binding: either they effect an initial transient binding, relaying substrate to a second site on the Dl tier where unfolding ensues, or they may serve as an entropic brush to clear the latter site of non-specifically bound ligands or substrates bound in non-productive complexes.     

Studies on the compaction of isolated nucleoids from Escherichia coli

The genomic DNA of Escherichia coli is contained in one or two compact bodies known as nucleoids. Isolation of typically shaped nucleoids requires control of DNA expansion, accomplished here by a modification of the polylysine-spermidine procedure. The ability to control expansion of in vitro nucleoids has application in nucleoid purification and in preparation of samples for high-resolution imaging, and may allow an increased resolution in gene localization studies. Polylysine of relatively low average molecular weight (approximately 3 kDa) is used to produce lysates containing nucleoids that are several-fold expanded relative to the sizes of in vivo nucleoids. These expanded forms can be converted to compact forms similar in dimensions to the cellular nucleoids by either a further addition of polylysine or by incubation of diluted lysates at 37 degrees C. The incubation at 37 degrees C is accompanied by autolytic degradation of most ribosomal RNA. Hyperchromism and circular dichroism spectra indicate that polylysine-DNA complexes are modified during the incubation. Compact forms of the nucleoid can be progressively reexpanded by exposure to salt solutions. Nucleoid compaction was similar in lysates made from rapidly or slowly growing cells or from cells that had been briefly treated with chloramphenicol to reduce linkages between DNA and cell envelope.     

Relationship between two proprioceptive measures and stiffness at the ankle.

Previous research has investigated the role of proprioception and stiffness in the control of joint stability. However, to date, no research has been done on the relationship between proprioception and stiffness. Therefore, the purpose of this study was to determine the relationship between force sense, joint reposition sense, and stiffness at the ankle. A heterogeneous sample was obtained for this study; 20 of the 40 participants had a history of ankle sprains, and 13 of the 20 had been diagnosed by a physician (two mild ankle sprains, seven moderate sprains, four severe sprains). All subjects were asymptomatic and active at the time of the study. Active joint reposition sense was measured using a custom-built ankle goniometer, force sense was measured unilaterally and contralaterally with a load cell, and ankle muscle stiffness was measured via transient oscillation using a custom-built inversion-eversion cradle. We found no significant correlations between stiffness and joint reposition sense, with values of r ranging from 0.01 to 0.21. Significant correlations were found between stiffness and force sense. Specifically, contralateral force sense reproduction was significantly correlated to stiffness in the injured or "involved" ankle (r's ranging from 0.47 to 0.65; P< or =0.008). Whether the decreased ability to appropriately sense force (increased error) sends information to the central nervous system to increase muscle stiffness in response to an unexpected loss of stability, or whether these two phenomena function independently and both change concurrently as a result of injury to the system requires further investigation.     

MesA, a novel fungal protein required for the stabilization of polarity axes in Aspergillus nidulans

The Aspergillus nidulans proteome possesses a single formin, SepA, which is required for actin ring formation at septation sites and also plays a role in polarized morphogenesis. Previous observations imply that complex regulatory mechanisms control the function of SepA and ensure its correct localization within hyphal tip cells. To characterize these mechanisms, we undertook a screen for mutations that enhance sepA defects. Of the mutants recovered, mesA1 causes the most dramatic defect in polarity establishment when SepA function is compromised. In a wild-type background, mesA1 mutants undergo aberrant hyphal morphogenesis, whereas septum formation remains unaffected. Molecular characterization revealed that MesA is a novel fungal protein that contains predicted transmembrane domains and localizes to hyphal tips. We show that MesA promotes the localized assembly of actin cables at polarization sites by facilitating the stable recruitment of SepA. We also provide evidence that MesA may regulate the formation or distribution of sterol-rich membrane domains. Our results suggest that these domains may be part of novel mechanism that directs SepA to hyphal tips.     

Unexpected foraminiferal diversity revealed by small-subunit rDNA analysis of Antarctic sediment

Studies of benthic Foraminifera typically rely on the morphological identification of dried specimens. This approach can introduce sampling bias against small, delicate, or morphologically ambiguous forms. To overcome this limitation, we extracted total DNA from sediment followed by PCR using group- and species-specific primers. Phylogenetic analyses revealed that approximately ninety percent of the PCR products represented previously undescribed sequence types that group with undersampled members of the allogromiid Foraminifera. We also used a modification of this technique to track individual species in sediment fractions too fine for normal morphological identification, and to confirm species placement of morphologically ambiguous foraminiferans. We were able to identify the DNA of several large foraminiferal species in fine fractions in a seasonally-dependent manner, indicating that in some seasons the majority of the standing stock of these species exists as gametes/juveniles. The approach outlined here represents a powerful strategy for exploring the total diversity of benthic foraminiferal communities.