Rescue of DeltaF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in cftr, a gene encoding a PKA-regulated Cl(-) channel. The most common mutation results in a deletion of phenylalanine at position 508 (DeltaF508-CFTR) that impairs protein folding, trafficking, and channel gating in epithelial cells. In the airway, these defects alter salt and fluid transport, leading to chronic infection, inflammation, and loss of lung function. There are no drugs that specifically target mutant CFTR, and optimal treatment of CF may require repair of both the folding and gating defects. Here, we describe two classes of novel, potent small molecules identified from screening compound libraries that restore the function of DeltaF508-CFTR in both recombinant cells and cultures of human bronchial epithelia isolated from CF patients. The first class partially corrects the trafficking defect by facilitating exit from the endoplasmic reticulum and restores DeltaF508-CFTR-mediated Cl(-) transport to more than 10% of that observed in non-CF human bronchial epithelial cultures, a level expected to result in a clinical benefit in CF patients. The second class of compounds potentiates cAMP-mediated gating of DeltaF508-CFTR and achieves single-channel activity similar to wild-type CFTR. The CFTR-activating effects of the two mechanisms are additive and support the rationale of a drug discovery strategy based on rescue of the basic genetic defect responsible for CF.     

Analysis of the individual contributions of Igalpha (CD79a)- and Igbeta (CD79b)-mediated tonic signaling for bone marrow B cell development and peripheral B cell maturation

The individual contribution of Igalpha and Igbeta for BCR-triggered fates is unclear. Prior evidence supports conflicting ideas concerning unique as well as redundant functions for these proteins in the context of BCR/pre-BCR signaling. Part of this ambiguity may reflect the recent appreciation that Igalpha and Igbeta participate in both Ag-independent (tonic) and Ag-dependent signaling. The present study undertook defining the individual requirement for Igalpha and Igbeta under conditions where only ligand-independent tonic signaling was operative. In this regard, we have constructed chimeric proteins containing one or two copies of the cytoplasmic domains of either Igalpha or Igbeta and Igalpha/Igbeta heterodimers with targeted Tyr-->Phe modifications. The ability of these proteins to act as surrogate receptors and trigger early bone marrow and peripheral B cell maturation was tested in RAG2(-/-) primary pro-B cell lines and in gene transfer experiments in the muMT mouse model. We considered that the threshold for a functional activity mediated by the pre-BCR/BCR might only be reached when two functional copies of the Igalpha/Igbeta ITAM domain are expressed together, and therefore the specificity conferred by these proteins can only be observed in these conditions. We found that the ligand-independent tonic signal is sufficient to drive development into mature follicular B cells and both Igalpha and Igbeta chains supported formation of this population. In contrast, neither marginal zone nor B1 mature B cell subsets develop from bone marrow precursors under conditions where only tonic signals are generated.     

Biochemical and genetic characterization of an early step in a novel pathway for the biosynthesis of aromatic amino acids and p-aminobenzoic acid in the archaeon Methanococcus maripaludis

Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon and facultative autotroph capable of biosynthesizing all the amino acids and vitamins required for growth. In this work, the novel 6-deoxy-5-ketofructose-1-phosphate (DKFP) pathway for the biosynthesis of aromatic amino acids (AroAAs) and p-aminobenzoic acid (PABA) was demonstrated in M. maripaludis. Moreover, PABA was shown to be derived from an early intermediate in AroAA biosynthesis and not from chorismate. Following metabolic labelling with [U-(13)C]-acetate, the expected enrichments for phenylalanine and arylamine derived from PABA were observed. DKFP pathway activity was reduced following growth with aryl acids, an alternative source of the AroAAs. Lastly, a deletion mutant of aroA', which encodes the first step in the DKFP pathway, required AroAAs and PABA for growth. Complementation of the mutants by an aroA' expression vector restored the wild-type phenotype. In contrast, a deletion of aroB', which encodes the second step in the DKFP pathway, did not require AroAAs or PABA for growth. Presumably, methanococci contain an alternative activity for this step. These results identify the initial reactions of a new pathway for the biosynthesis of PABA in methanococci.     

Characterization of an experimental miniature bioreactor for cellular perturbation studies

A mini bioreactor (3.0 mL volume) has been developed and shown to be a versatile tool for rapidly screening and quantifying the response of organisms on environmental perturbations. The mini bioreactor is essentially a plug flow device transformed into a well-mixed reactor by a recycle flow of the broth. The gas and liquid phases are separated by a silicone membrane. Dynamic mass transfer experiments were performed to determine the mass transfer capacities for oxygen and carbon dioxide. The mass transfer coefficients for oxygen and carbon dioxide were found to be 1.55 +/- 0.17 x 10(-5) m/s and 4.52 +/- 0.60 x 10(-6) m/s, respectively. Cultivation experiments with the 3.0 mL bioreactor show that (i) it can maintain biomass in the same physiological state as the 4.0 L lab scale bioreactor, (ii) reproducible perturbation experiments such as changing substrate uptake rate can be readily performed and the physiological response monitored quantitatively in terms of the O2 and CO2 uptake and production rates.     

Global organization and function of mammalian cytosolic proteasome pools: Implications for PA28 and 19S regulatory complexes

Proteolytic activity of the 20S proteasome is regulated by activators that govern substrate movement into and out of the catalytic chamber. However, the physiological relationship between activators, and hence the relative role of different proteasome species, remains poorly understood. To address this problem, we characterized the total pool of cytosolic proteasomes in intact and functional form using a single-step method that bypasses the need for antibodies, proteasome modification, or column purification. Two-dimensional Blue Native(BN)/SDS-PAGE and tandem mass spectrometry simultaneously identified six native proteasome populations in untreated cytosol: 20S, singly and doubly PA28-capped, singly 19S-capped, hybrid, and doubly 19S-capped proteasomes. All proteasome species were highly dynamic as evidenced by recruitment and exchange of regulatory caps. In particular, proteasome inhibition with MG132 markedly stimulated PA28 binding to exposed 20S alpha-subunits and generated doubly PA28-capped and hybrid proteasomes. PA28 recruitment virtually eliminated free 20S particles and was blocked by ATP depletion. Moreover, inhibited proteasomes remained stably associated with distinct cohorts of partially degraded fragments derived from cytosolic and ER substrates. These data establish a versatile platform for analyzing substrate-specific proteasome function and indicate that PA28 and 19S activators cooperatively regulate global protein turnover while functioning at different stages of the degradation cycle.     

Reverse translocation of tRNA in the ribosome

A widely held view is that directional movement of tRNA in the ribosome is determined by an intrinsic mechanism and driven thermodynamically by transpeptidation. Here, we show that, in certain ribosomal complexes, the pretranslocation (PRE) state is thermodynamically favored over the posttranslocation (POST) state. Spontaneous and efficient conversion from the POST to PRE state is observed when EF-G is depleted from ribosomes in the POST state or when tRNA is added to the E site of ribosomes containing P-site tRNA. In the latter assay, the rate of tRNA movement is increased by streptomycin and neomycin, decreased by tetracycline, and not affected by the acylation state of the tRNA. In one case, we provide evidence that complex conversion occurs by reverse translocation (i.e., direct movement of the tRNAs from the E and P sites to the P and A sites, respectively). These findings have important implications for the energetics of translocation.     

Triacylglycerol-rich lipoproteins protect lipoprotein lipase from inactivation by ANGPTL3 and ANGPTL4

Lipoprotein lipase (LPL) is important for clearance of triacylglycerols (TG) from plasma both as an enzyme and as a bridging factor between lipoproteins and receptors for endocytosis. The amount of LPL at the luminal side of the capillary endothelium determines to what extent lipids are taken up. Mechanisms to control both the activity of LPL and its transport to the endothelial sites are regulated, but poorly understood. Angiopoietin-like proteins (ANGPTLs) 3 and 4 are potential control proteins for LPL, but plasma concentrations of ANGPTLs do not correlate with plasma TG levels. We investigated the effects of recombinant human N-terminal (NT) ANGPTLs3 and 4 on LPL-mediated bridging of TG-rich lipoproteins to primary mouse hepatocytes and found that the NT-ANGPTLs, in concentrations sufficient to cause inactivation of LPL in vitro, were unable to prevent LPL-mediated lipoprotein uptake. We therefore investigated the effects of lipoproteins (chylomicrons, VLDL and LDL) on the inactivation of LPL in vitro by NT-ANGPTLs3 and 4 and found that LPL activity was protected by TG-rich lipoproteins. In vivo, postprandial TG protected LPL from inactivation by recombinant NT-ANGPTL4 injected to mice. We conclude that lipoprotein-bound LPL is stabilized against inactivation by ANGPTLs. The levels of ANGPTLs found in blood may not be sufficient to overcome this stabilization. Therefore it is likely that the prime site of action of ANGPTLs on LPL is in subendothelial compartments where TG-rich lipoprotein concentration is lower than in blood. This could explain why the plasma levels of TG and ANGPTLs do not correlate.     

Spatial ecology of wolverines at the southern periphery of distribution

Wolverines (Gulo gulo) in the conterminous United States have experienced range contraction, are uncommon, and have been designated as warranted for protection under the United States Endangered Species Act. Data from the southern edge of the wolverine's circumpolar distribution is sparse, and development of effective conservation strategies would benefit from a more complete understanding of the species' ecology. We captured and radio-monitored 30 wolverines in the Greater Yellowstone Ecosystem (GYE), tested for seasonal habitat selection by elevation band, and examined a suite of spatial characteristics to clarify our understanding of the wolverine's niche. Wolverines in GYE selected for areas >2,600 m latitude-adjusted elevation (LAE; n = 2,257 wolverine locations [12 F, 6 M]). Wolverines avoided areas <2,150 m LAE, including during winter when the vast majority of ungulates are pushed to these elevations by deep snow. Wolverine home ranges were large relative to body size, averaging 303 km² for adult females and 797 km² for adult males (n = 13 [8 F, 5 M] and 33 wolverine-years). Resident adults fit with Global Positioning System (GPS) collars used an area >75% the size of their multi-year home range in an average of 32 days (n = 7 [5 F, 2 M]). Average movement rates of 1.3 km/2-hr indicated that both sexes move distances equivalent to the diameter of their home range every 2 days or the circumference of their home range in <1 week (n = 1,329 2-hr movements, n = 12 individuals [7 F, 5 M]). This capability for movement, the short time-frame over which home ranges were developed, and a lack of home range overlap by same sex adults ( , 90% CI = 0.0–4.8%, n = 22 pairs) suggested territoriality. We estimated wolverine density to be 3.5/1,000 km² of area >2,150 m LAE (95% CI = 2.8–9.6). Dispersal movements extended to at least 170 km for both sexes (n = 5 F, 2 M). At the southern edge of distribution, where suitable and unsuitable conditions exist in close proximity, wolverines selected high-elevation areas near alpine tree-line where a mix of forest, meadow, and boulder fields were present, deep snow-cover existed during winter, and low temperatures near freezing can occur throughout the year. Persistence in these areas where the growing season is brief requires large home ranges that are regularly patrolled, a social system that provides exclusive access to resources, and low densities. These characteristics, along with low reproductive rates, are prevalent throughout the species range, indicating that wolverines are specialists at exploiting a cold, unproductive niche where interspecific competition is limited. The vulnerability inherent in occupying this unproductive niche was likely influential in previous declines within the conterminous United States and will remain a factor as wolverines encounter modern human influences. Conserving wolverines in the conterminous United States will require collaborative management over a large geographic scale. © 2011 The Wildlife Society.     

HNF1B and endometrial cancer risk: results from the PAGE study

We examined the association between HNF1B variants identified in a recent genome-wide association study and endometrial cancer in two large case-control studies nested in prospective cohorts: the Multiethnic Cohort Study (MEC) and the Women's Health Initiative (WHI) as part of the Population Architecture using Genomics and Epidemiology (PAGE) study. A total of 1,357 incident cases of invasive endometrial cancer and 7,609 controls were included in the analysis (MEC: 426 cases/3,854 controls; WHI: 931 cases/3,755 controls). The majority of women in the WHI were European American, while the MEC included sizable numbers of African Americans, Japanese and Latinos. We estimated the odds ratios (ORs) per allele and 95% confidence intervals (CIs) of each SNP using unconditional logistic regression adjusting for age, body mass index, and four principal components of ancestry informative markers. The combined ORs were estimated using fixed effect models. Rs4430796 and rs7501939 were associated with endometrial cancer risk in MEC and WHI with no heterogeneity observed across racial/ethnic groups (P ≥ 0.21) or between studies (P ≥ 0.70). The OR(per allele) was 0.82 (95% CI: 0.75, 0.89; P = 5.63 × 10(-6)) for rs4430796 (G allele) and 0.79 (95% CI: 0.73, 0.87; P = 3.77 × 10(-7)) for rs7501939 (A allele). The associations with the risk of Type I and Type II tumors were similar (P ≥ 0.19). Adjustment for additional endometrial cancer risk factors such as parity, oral contraceptive use, menopausal hormone use, and smoking status had little effect on the results. In conclusion, HNF1B SNPs are associated with risk of endometrial cancer and that the associated relative risks are similar for Type I and Type II tumors.