Rab11b Regulates the Apical Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator in Polarized Intestinal Epithelial Cells

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, ΔF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.     

HspBP1 levels are elevated in breast tumor tissue and inversely related to tumor aggressiveness

HspBP1 is a co-chaperone that binds to and regulates the chaperone Hsp70 (Hsp70 is used to refer to HSPA1A and HSPA1B). Hsp70 is known to be elevated in breast tumor tissue, therefore the purpose of these studies was to quantify the expression of HspBP1 in primary breast tumors and in serum of these patients with a follow-up analysis after 6 to 7 years. Levels of HspBP1, Hsp70, and anti-HspBP1 antibodies in sera of breast cancer patients and healthy individuals were measured by enzyme-linked immunosorbent assay. Expression of HspBP1 was quantified from biopsies of tumor and normal breast tissue by Western blot analysis. The data obtained were analyzed for association with tumor aggressiveness markers and with patient outcome. The levels of HspBP1 and Hsp70 were significantly higher in sera of patients compared to sera of healthy individuals. HspBP1 antibodies did not differ significantly between groups. HspBP1 levels were significantly higher in tumor (14.46 ng/μg protein, n = 51) compared to normal adjacent tissue (3.17 ng/μg protein, n = 41, p < 0.001). Expression of HspBP1 was significantly lower in patients with lymph node metastasis and positive for estrogen receptors. HspBP1 levels were also significantly lower in patients with a higher incidence of metastasis and death following a 6 to 7-year follow-up. The HspBP1/Hsp70 molar ratio was not associated with the prognostic markers analyzed. Our results indicate that low HspBP1 expression could be a candidate tumor aggressiveness marker. This work was supported by FAPERGS, CNPq, and the National Institute of General Medical Sciences grant number GM072628-02 (V.G.) The expression of HspBP1 (an Hsp70 co-chaperone) was analyzed in tumor samples and sera from breast cancer patients. HspBP1 is over expressed in these tumors and a seven year follow-up analysis found an association with a poor prognosis. Chaperones have been shown to play important roles in tumor biology and immunology; therefore, we believe the data in this study will serve as a basis for the formulation of a new hypothesis on chaperone-co-chaperone interactions and their role in tumor growth.     

Crucial role for LKB1 to AMPKα2 axis in the regulation of CD36-mediated long-chain fatty acid uptake into cardiomyocytes

Enhanced contractile activity increases cardiac long-chain fatty acid (LCFA) uptake via translocation of CD36 to the sarcolemma, similarly to increase in glucose uptake via GLUT4 translocation. AMP-activated protein kinase (AMPK) is assumed to mediate contraction-induced LCFA utilization. However, which catalytic isoform (AMPKα1 versus AMPKα2) is involved, is unknown. Furthermore, no studies have been performed on the role of LKB1, a kinase with AMPKK activity, on the regulation of cardiac LCFA utilization. Using different mouse models (AMPKα2-kinase-dead, AMPKα2-knockout and LKB1-knockout mice), we tested whether LKB1 and/or AMPK are required for stimulation of LCFA and glucose utilization upon treatment of cardiomyocytes with compounds (oligomycin/AICAR/dipyridamole) which induce CD36 translocation similar to that seen upon contraction. In AMPKα2- kinase-dead cardiomyocytes, the stimulating effects of oligomycin and AICAR on palmitate and deoxyglucose uptake and palmitate oxidation were almost completely lost. Moreover, in AMPKα2- and LKB1-knockout cardiomyocytes, oligomycin-induced LCFA and deoxyglucose uptake were completely abolished. However, the stimulatory effect of dipyridamole on palmitate uptake and oxidation was preserved in AMPKα2-kinase-dead cardiomyocytes. In conclusion, in the heart there is a signaling axis consisting of LKB1 and AMPKα2 which activation results in enhanced LCFA utilization, similarly to enhanced glucose uptake. In addition, an unknown dipyridamole-activated pathway can stimulate cardiac LCFA utilization by activating signaling components downstream of AMPK.     

Thermus thermophilus as biological model

Thermus spp is one of the most wide spread genuses of thermophilic bacteria, with isolates found in natural as well as in man-made thermal environments. The high growth rates, cell yields of the cultures, and the constitutive expression of an impressively efficient natural competence apparatus, amongst other properties, make some strains of the genus excellent laboratory models to study the molecular basis of thermophilia. These properties, together with the fact that enzymes and protein complexes from extremophiles are easier to crystallize have led to the development of an ongoing structural biology program dedicated to T. thermophilus HB8, making this organism probably the best so far known from a protein structure point view. Furthermore, the availability of plasmids and up to four thermostable antibiotic selection markers allows its use in physiological studies as a model for ancient bacteria. Regarding biotechnological applications this genus continues to be a source of thermophilic enzymes of great biotechnological interest and, more recently, a tool for the over-expression of thermophilic enzymes or for the selection of thermostable mutants from mesophilic proteins by directed evolution. In this article, we review the properties of this organism as biological model and its biotechnological applications.     

A climatic and taxonomic comparison between leaf litter and standing vegetation from a Florida swamp woodland

One method to determine past climate has been the use of leaf morphological characteristics of fossil leaves quantified using modern climate and canopy leaf characteristics. Fossil assemblages are composed of abscised leaves, and climate may be more accurately determined by using leaves from leaf litter instead of the canopy. To better understand whether taphonomic processes make a difference in this relationship, a north-central Florida woodland was sampled to determine the morphologically based climate estimates from these leaves. Leaves from woody, dicotyledonous plants were collected and identified, then compared using presence/absence data and analyzed using several linear regression equations and the CLAMP data set. Although the majority of standing vegetation was reflected in leaf litter, some inconsistencies were observed, which may reflect plant community structure or sampling technique. Mean annual temperature (MAT) and growing season precipitation (GSP) were estimated from leaf litter morphological characters and living leaves. Overall, values for MAT estimated from litter and living leaves were cooler than actual MATs, although several accurate and high estimates were obtained depending on the predictive method used. Estimated GSP values were higher than actual GSPs. Statistically, no difference was observed between MAT and GSP estimates derived from leaf litter vs. estimates derived from living leaves, with one exception.     

Use of magnetic carboxyl beads to purify a cationic peptide in a batch system

Antimicrobial peptides (AMPs) are cationic molecules that are good leads for new antiinfective drugs. To obtain sufficient amounts, recombinant AMPs are generally produced as fusion proteins in Escherichia coli. Fusion partners facilitate purification of recombinant proteins. Fusion proteins are then cleaved by specific proteases, and cationic peptides are purified by size exclusion chromatography or ion exchange chromatography, neither of which is easily applicable to small volumes of diluted peptide samples. We developed a small-scale system that is easily adaptable for high-throughput screening and uses carboxyl magnetic beads to purify a cationic peptide from its fusion partner.     

Phylogeography of the common vampire bat (Desmodus rotundus): Marked population structure, Neotropical Pleistocene vicariance and incongruence between nuclear and mtDNA markers

Background  

The common vampire bat Desmodus rotundus is an excellent model organism for studying ecological vicariance in the Neotropics due to its broad geographic range and its preference for forested areas as roosting sites. With the objective of testing for Pleistocene ecological vicariance, we sequenced a mitocondrial DNA (mtDNA) marker and two nuclear markers (RAG2 and DRB) to try to understand how Pleistocene glaciations affected the distribution of intraspecific lineages in this bat.