Characterization of a panel of six β2-adrenergic receptor antibodies by indirect immunofluorescence microscopy

Background

The β₂-adrenergic receptor (β₂AR) is a primary target for medications used to treat asthma. Due to the low abundance of β₂AR, very few studies have reported its localization in tissues. However, the intracellular location of β₂AR in lung tissue, especially in airway smooth muscle cells, is very likely to have a significant impact on how the airways respond to β-agonist medications. Thus, a method for visualizing β₂AR in tissues would be of utility. The purpose of this study was to develop an immunofluorescent labeling technique for localizing native and recombinant β₂AR in primary cell cultures.

Methods

A panel of six different antibodies were evaluated in indirect immunofluorescence assays for their ability to recognize human and rat β₂AR expressed in HEK 293 cells. Antibodies capable of recognizing rat β₂AR were identified and used to localize native β₂AR in primary cultures of rat airway smooth muscle and epithelial cells. β₂AR expression was confirmed by performing ligand binding assays using the β-adrenergic antagonist [3H] dihydroalprenolol ^([3H]DHA).

Results

Among the six antibodies tested, we identified three of interest. An antibody developed against the C-terminal 15 amino acids of the human β₂AR (Ab-Bethyl) specifically recognized human but not rat β₂AR. An antibody developed against the C-terminal domain of the mouse β₂AR (Ab-sc570) specifically recognized rat but not human β₂AR. An antibody developed against 78 amino acids of the C-terminus of the human β₂AR (Ab-13989) was capable of recognizing both rat and human β₂ARs. In HEK 293 cells, the receptors were predominantly localized to the cell surface. By contrast, about half of the native rat β₂AR that we visualized in primary cultures of rat airway epithelial and smooth muscle cells using Ab-sc570 and Ab-13989 was found inside cells rather than on their surface.

Conclusion

Antibodies have been identified that recognize human β₂AR, rat β₂AR or both rat and human β₂AR. Interestingly, the pattern of expression in transfected cells expressing millions of receptors was dramatically different from that in primary cell cultures expressing only a few thousand native receptors. We anticipate that these antibodies will provide a valuable tool for evaluating the expression and trafficking of β₂AR in tissues.     

New polyphenols active on beta-amyloid aggregation

New polyphenol classes have been tested against amyloid-beta peptide aggregation. We have identified four novel polyphenols which could be efficient fibril inhibitors in Alzheimer's disease: malvidin and its glucoside and curculigosides B and D. We suggest that molecules with the particular C(6)-linkers-C(6) structure could be potent inhibitors. From the results reported for the flavan-3-ol family, their anti-amyloidogenic effects against whole peptides (1-40 and 1-42) could involve several binding sites.     

Morphological evolution of the lizard skull: a geometric morphometrics survey

Patterns of diversity among lizard skulls were studied from a morphological, phylogenetic, and functional perspective. A sample of 1,030 lizard skulls from 441 species in 17 families was used to create a lizard skull morphospace. This morphospace was combined with a phylogeny of lizard families to summarize general trends in the evolution of the lizard skull. A basal morphological split between the Iguania and Scleroglossa was observed. Iguanians are characterized by a short, high skull, with large areas of attachment for the external adductor musculature, relative to their sister group. The families of the Iguania appear to possess more intrafamilial morphological diversity than families of the Scleroglossa, but rarefaction of the data reveals this to be an artifact caused by the greater number of species represented in Iguanian families. Iguanian families also appear more dissimilar to one another than families of the Scleroglossa. Permutation tests indicate that this pattern is real and not due to the smaller number of families in the Iguanidae. Parallel and convergent evolution is observed among lizards with similar diets: ant and termite specialists, carnivores, and herbivores. However, these patterns are superimposed over the more general phylogenetic pattern of lizard skull diversity. This study has three central conclusions. Different clades of lizards show different patterns of disparity and divergence in patterns of morphospace occupation. Phylogeny imposes a primary signal upon which a secondary ecological signal is imprinted. Evolutionary patterns in skull metrics, taken with functional landmarks, allow testing of trends and the development of new hypotheses concerning both shape and biomechanics.     

Purinergic-induced signaling in C11-MDCK cells inhibits the secretory Na-K-Cl cotransporter

Purinergic inhibition of Na-K-Cl cotransport has been noted in various renal epithelial cells derived from the collecting tubule, including Madin-Darby canine kidney (MDCK) cells. In recent studies, we have observed purinergic inhibition of Na-K-Cl cotransport in C11-MDCK subclones (alpha-intercalated-like cells). Interestingly, Na-K-Cl cotransport activity was also detected in C7-MDCK subclones (principal-like cells) but was not affected by ATP. In this investigation, we have transfected the human Na-K-Cl cotransporter (huNKCC1) in both C11 and C7 cells to determine whether these differences in NKCC regulation by ATP were due to cell-specific purinoceptor signaling pathways or to cell-specific isoforms/splice variants of the transporter. In both cell lines, we found that endogenous as well as huNKCC1-derived cotransport activity was restricted to the basolateral side. In addition, we were able to show that extracellular application of 100 microM ATP or 100 microM UTP abolished NKCC activity in both mock- and huNKCC1-transfected C11 cells but not in mock- and huNKCC1-transfected C7 cells; in C11 cells, intriguingly, this inhibition was not affected by inhibitors of RNA and protein synthesis and occurred even though expression levels of UTP-sensitive P2Y2-, P2Y4-, and P2Y6-purinoceptors were not different from those observed in C7 cells. These results suggest that C11 cells express an undetermined type of UTP-sensitive P2-purinoceptors or a unique P2Y-purinoceptor-triggered signaling cascade that leads to inhibition of NKCC1.     

A recA-LexA-dependent pathway mediates ciprofloxacin-induced fibronectin binding in Staphylococcus aureus

Subinhibitory concentrations of ciprofloxacin (CPX) raise the fibronectin-mediated attachment of fluoroquinolone-resistant Staphylococcus aureus by selectively inducing fnbB coding for one of two fibronectin-binding proteins: FnBPB. To identify candidate regulatory pathway(s) linking drug exposure to up-regulation of fnbB, we disrupted the global response regulators agr, sarA, and recA in the highly quinolone-resistant strain RA1. Whereas agr and sarA mutants of RA1 exposed to CPX still displayed increased adhesion to fibronectin, the CPX-triggered response was abolished in the uvs-568 recA mutant, but was restored following complementation with wild type recA. Steady-state levels of recA and fnbB, but not fnbA, mRNA were co-coordinately increased >3-fold in CPX-exposed strain RA1. Electrophoretic mobility shift assays revealed specific binding of purified S. aureus SOS-repressor LexA to recA and fnbB, but not to fnbA or rpoB promoters. DNase I footprint analysis showed LexA binding overlapping the core promoter elements in fnbB. We conclude that activation of recA and derepression of lexA-regulated genes by CPX may represent a response to drug-induced damage that results in a novel induction of a virulence factor leading to increased bacterial tissue adherence.     

Exploiting type 3 complement receptor for TNF-alpha suppression, immune evasion, and progressive pulmonary fungal infection

TNF-alpha is crucial in defense against intracellular microbes. Host immune cells use type 3 complement receptors (CR3) to regulate excess TNF-alpha production during physiological clearance of apoptotic cells. BAD1, a virulence factor of Blastomyces dermatitidis, is displayed on yeast and released during infection. BAD1 binds yeast to macrophages (Mphi) via CR3 and CD14 and suppresses TNF-alpha, which is required for resistance. We investigated whether blastomyces adhesin 1 (BAD1) exploits host receptors for immune deviation and pathogen survival. Soluble BAD1 rapidly entered Mphi, accumulated intracellularly by 10 min after introduction to cells, and trafficked to early and late endosomes. Inhibition of receptor recycling by monodansyl cadaverine blocked association of BAD1 with Mphi and reversed TNF-alpha suppression in vitro. Inhibition of BAD1 uptake with cytochalasin D and FcR-redirected delivery of soluble BAD1 as Ag-Ab complexes or of wild-type yeast opsonized with IgG similarly reversed TNF-alpha suppression. Hence, receptor-mediated entry of BAD1 is requisite in TNF-alpha suppression, and the route of entry is critical. Binding of soluble BAD1 to Mphi of CR3(-/-) and CD14(-/-) mice was reduced to 50 and 33%, respectively, of that in wild-type mice. Mphi of CR3(-/-) and CD14(-/-) mice resisted soluble BAD1 TNF-alpha suppression in vitro, but, in contrast to CR3(-/-) cells, CD14(-/-) cells were still subject to suppression mediated by surface BAD1 on wild-type yeast. CR3(-/-) mice resisted both infection and TNF-alpha suppression in vivo, in contrast to wild-type and CD14(-/-) mice. BAD1 of B. dermatitidis thus co-opts normal host cell physiology by exploiting CR3 to subdue TNF-alpha production and foster pathogen survival.     

The first alpha helix of Bax plays a necessary role in its ligand-induced activation by the BH3-only proteins Bid and PUMA

The mechanism by which some BH3-only proteins of the Bcl-2 family directly activate the "multidomain" proapoptotic member Bax is poorly characterized. We report that the first alpha helix (Halpha1) of Bax specifically interacts with the BH3 domains of Bid and PUMA but not with that of Bad. Inhibition of this interaction, by a peptide comprising Halpha1 or by a mutation in this helix, prevents ligand-induced activation of Bax by Bid, PUMA, or their BH3 peptides. Halpha1-mutated Bax, which can mediate death induced by Bad or its BH3 peptide, does not mediate that induced by Bid, PUMA, or their BH3 peptides. The response of Halpha1-mutated Bax to Bid can be restored by a compensating mutation in Bid BH3. Thus, a specific interaction between Bax Halpha1 and their BH3 domains allows Bid and PUMA to function as "death agonists" of Bax, whereas Bad recruits Bax activity through a distinct pathway.     

Conserved CDR3 regions in T-cell receptor (TCR) CD8(+) T cells that recognize the Tax11-19/HLA-A*0201 complex in a subject infected with human T-cell leukemia virus type 1: relationship of T-cell fine specificity and major histocompatibility complex/peptide/TCR crystal structure

We investigated the T-cell receptor (TCR) repertoire of CD8(+) T cells that recognize the Tax11-19 immunodominant epitope of Tax protein expressed by human T-cell leukemia virus (HTLV-1) that is implicated in the disease HTLV-1-associated myelopathy (HAM/TSP). A panel of Tax11-19-reactive CD8(+) T-cell clones was generated by single-cell cloning of Tax11-19/HLA-A*0201 tetramer-positive peripheral blood lymphocytes from an HTLV-1-infected individual. The analyses of TCR usage revealed that the combination of diverse TCR alpha and beta chains could be used for the recognition of Tax11-19 but the major population of T-cell clones (15 of 24 clones) expressed the TCR V beta 13S1 and V alpha 17 chain. We found striking similarities in CDR3 regions of TCR alpha and beta chains between our major group of CD8(+) T-cell clones and those originating from different subjects as previously reported, including TCRs with resolved crystal structures. A 3-amino-acid sequence (PG-G) in the CDR3 region of the V beta chain was conserved among all the Tax11-19-reactive T-cell clones expressing V beta 13S1 and V alpha 17 chains. Conserved amino acids in the CDR3 region do not directly contact the Tax11-19 peptide, as corroborated by the crystal structure of B7-TCR, a TCR that is almost identical to VB13S1 clones isolated in this study. Analysis of fine peptide specificity using altered peptide ligands (APL) of Tax11-19 revealed a similar recognition pattern among this panel of T-cell clones. These data suggest that the PG-G amino acids in the CDR3 beta loop provide a structural framework necessary for the maintenance of the tertiary TCR structure.     

Clindamycin suppresses virulence expression in inducible clindamycin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> strains

Clindamycin is a protein synthesis inhibitory agent that has the ability to suppress the expression of virulence factors in Staphylococcus aureus. Recent guidelines recommend the use of clindamycin for the treatment of toxin-mediated infections. Clindamycin modulates virulence expression at sub-inhibitory concentrations (sub-MICs) in clindamycin-susceptible S. aureus strains but previous report shown that this effect was supressed for constitutive clindamycin resistant strains. However, no data are currently available on the impact of clindamycin at sub-MICs on the virulence of inducible clindamycin-resistant S. aureus strains. Here, we show that sub-MICs of clindamycin decrease Panton–Valentine leucocidin, toxic-shock-staphylococcal toxin (TSST-1) and alpha-haemolysin (Hla) expression in six inducible clindamycin-resistant isolates cultivated in vitro in CCY medium. These results suggest that the clindamycin anti-toxin effect is retained for inducible clindamycin-resistant S. aureus isolates; therefore, its usage should be considered within the treatment regimen of toxin related infections for inducible clindamycin-resistant S. aureus.