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The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates electrolyte and fluid balance in epithelial tissues. While activation of CFTR is vital to treating cystic fibrosis, selective inhibition of CFTR is a potential therapeutic strategy for secretory diarrhea and autosomal dominant polycystic kidney disease. Although several CFTR inhibitors have been developed by high-throughput screening, their modes of action remain elusive. In this study, we determined the structure of CFTR in complex with the inhibitor CFTRinh-172 to an overall resolution of 2.7 Å by cryogenic electron microscopy. We observe that CFTRinh-172 binds inside the pore near transmembrane helix 8, a critical structural element that links adenosine triphosphate hydrolysis with channel gating. Binding of CFTRinh-172 stabilizes a conformation in which the chloride selectivity filter is collapsed, and the pore is blocked from the extracellular side of the membrane. Single-molecule fluorescence resonance energy transfer experiments indicate that CFTRinh-172 inhibits channel gating without compromising nucleotide-binding domain dimerization. Together, these data reconcile previous biophysical observations and provide a molecular basis for the activity of this widely used CFTR inhibitor. [ABSTRACT FROM AUTHOR]

The common pathogen Streptococcus pyogenes colonizes the human skin and tonsils and can invade underlying tissues. This requires the adhesion of S. pyogenes to host surface receptors mediated through adhesins. The laminin-binding protein Lbp has been suggested as an adhesin, specific for the human extracellular matrix protein laminin. Sequence alignments, however, indicate a relationship between Lbp and a family of bacterial metal-binding receptors. To further analyze the role of Lbp in S. pyogenes and its potential role in pathogenicity, Lbp has been crystallized, and its structure has been solved at a resolution of 2.45 [Angstrom] (R = 0.186; [R.sub.free] = 0.251). Lbp has the typical metal-binding receptor fold, comprising two globular [([beta]/[alpha]).sub.4] domains connected by a helical backbone. The two domains enclose the metal-binding site, which contains a zinc ion. The interaction of Lbp with laminin was further investigated and shown to be specific in vitro. Localization studies with antibodies specific for Lbp show that the protein is attached to the membrane. The data suggest that Lbp is primarily a zinc-binding protein, and we suggest that its interaction with laminin in vivo may be mediated via zinc bound to laminin.

Aminoglycoside-2'-phosphotransferase-IIa [APH(2')-IIa] is one of a number of homologous bacterial enzymes responsible for the deactivation of the aminoglycoside family of antibiotics and is thus a major component in bacterial resistance to these compounds. APH(2')-IIa produces resistance to several clinically important aminoglycosides (including kanamycin and gentamicin) in both gram-positive and gram-negative bacteria, most notably in Enterococcus species. We have determined the structures of two complexes of APH(2')-IIa, the binary gentamicin complex and a ternary complex containing adenosine-5'-([beta],[gamma]-methylene) triphosphate (AMPPCP) and streptomycin. This is the first crystal structure of a member of the APH(2') family of aminoglycoside phosphotransferases. The structure of the gentamicin-APH(2')-IIa complex was solved by multiwavelength anomalous diffraction methods from a single selenomethionine-substituted crystal and was refined to a crystallographic R factor of 0.210 ([R.sub.free], 0.271) at a resolution of 2.5 [Angstrom]. The structure of the AMPPCP-streptomycin complex was solved by molecular replacement using the gentamicin-APH(2')-IIa complex as the starting model. The enzyme has a two-domain structure with the substrate binding site located in a cleft in the C-terminal domain. Gentamicin binding is facilitated by a number of conserved acidic residues lining the binding cleft, with the A and B rings of the substrate forming the majority of the interactions. The inhibitor streptomycin, although binding in the same pocket as gentamicin, is orientated such that no potential phosphorylation sites are adjacent to the catalytic aspartate residue. The binding of gentamicin and streptomycin provides structural insights into the substrate selectivity of the APH(2') subfamily of aminoglycoside phosphotransferases, specifically, the selectivity between the 4,6-disubstituted and the 4,5-disubstituted aminoglycosides.

Principal describes how several arts-oriented initiatives helped improve at-risk student motivation and subsequent test scores at West Elementary School in Lancaster, Ohio. Initiatives included summer arts enrichment, integrated art and writing, coordinated music lessons, after-school tutoring, artistic discipline, schoolwide assemblies, and band classes. (PKP)

In Schizosaccharomyces pombe, the nuclear accumulation of Cdc25 peaks in G2 and is necessary for the proper timing of mitotic entry. Here, we identify the [sal3.sup.+] gene product as an importin-[beta] homolog that participates in the nuclear import of Cdc25. Loss of [sal3.sup.+] results in a cell cycle delay, failure to undergo G1 arrest under nitrogen-starvation conditions, and mislocalization of Cdc25 to the cytosol. Fusion of an exogenous classical nuclear localization sequence (cNLS) to Cdc25 restores its nuclear accumulation in a sal3 disruptant and suppresses the sal3 mutant phenotypes. In addition, we show that enhanced nuclear localization of Cdc25 at endogenous levels of expression advances the onset of mitosis. These results demonstrate that the nuclear translocation of Cdc25 is important for the timing of mitotic entry and that Sal3 plays an important role in this process.

Loss of the nonessential RNA-binding domain protein, Scw1, increases resistance to cell-wall-degrading enzymes in fission yeast. Surprisingly, scw1 null mutations also suppress the lethality of mutations (cdc11-136, cdc7-24, cdc14-118, sid1-239, sid2-250, sid3-106, sid4-A1, and mob1-1) at all levels of the sid pathway. This pathway forms part of the septation initiation network (SIN), which regulates the onset of septum formation and ensures the proper coupling of mitosis to cytokinesis. In contrast, scw[1.sup.-] mutations do not suppress ts alleles of the rng genes, cdc12 or cdc15. These mutations also prevent the formation of a septum and in addition block assembly and/or function of the contractile acto-myosin ring. sid mutants exhibit a hyper-sensitivity to cell-wall-degrading enzymes that is suppressed by loss of Scw1. Furthermore, scw[1.sup.-]-mediated rescue of sid mutants is abolished in the presence of calcofluor white, a compound that interferes with cell-wall synthesis. These data suggest that Scw1 acts in opposition to the SIN as a negative regulator of cell-wall/septum deposition. Unlike components of the SIN, Scw1 is predominantly a cytoplasmic protein and is not localized to the spindle pole body.

Mutational analysis was performed to identify amino acid residues that are necessary for the activity of 'Schizosaccharomyces pombe's Na+/H+ antiporter called sod2. Results show that the His residues of sod2 are important in the Na+/H+ exchanger of the fission yeast. These include His367, Asp241 and Asp266,267. The mutant H367R was unable to alkalinize the external medium and to remove sodium in an acidic medium.

The role of transforming growth factor (TGF)-beta1 in airway remodeling during asthma was analyzed in bovine airway smooth muscle cells (BASMC). Cultured BASMC exhibited enhanced hypertrophy and hyperplasia after treatment with TGF-beta1. TGF-beta1 also stimulated thymidine incorporation and increased the production of glycosaminoglycans by 68% in BASMC. Furthermore, the bimodal effects of the cytokine were mediated by the autocrine production of platelet-derived growth factor.

In the fission yeast, Schizosaccharomyces pombe, tolerance to high sodium and lithium concentrations requires the functioning of the sod2, [Na.sup.+]/[H.sup.+] antiporter. We have directly measured the activity of this antiporter and demonstrated reconstitution of the activity in gene deletion strains. In addition, we have shown that it can be transferred to, and its antiporter activity detected in, the budding yeast, Saccharomyces cerevisiae, where it also confers sodium and lithium tolerance. Proton flux through the S. pombe [Na.sup.+]/[H.sup.+] antiporter was directly measured using microphysiometry. The direction of transmembrane proton flux mediated by this antiporter was reversible, with protons being imported or exported in response to the external concentration of sodium. This bidirectional activity was also detected in S. cerevisiae strains expressing sod2 and expression of this gene complemented the sodium and lithium sensitivity resulting from inactivation of the ENA1/PMR2 encoded [Na.sup.+]-exporting AT-Pases. This suggests that antiporters or sodium pumps can be utilized interchangeably by S. cerevisiae to regulate internal sodium concentration. Potent inhibitors of mammalian [Na.sup.+]/[H.sup.+] exchangers were found to have no effect on sod2 activity. The proton flux mediated by sod2 was also found to be unaffected by perturbation of membrane potential or the plasma membrane proton gradient.

Quantum well structures, characterized before and after an epitaxial lift-off (ELO), reveal steady enhancement in F(T) of 12 to 20% and low frequency enhancement in G(max) of 1 to 2 dB in the ELO transistors. The enhancement is determined by the S parameters. An evaluation of the ELO structures indicates an enhancement in the carrier confinement after ELO produces an increase in transconductance. The effects of the transistor lift-off on an insulating substrate are predicted by the estimation of the ELO process for the two primary HEMT structures.

Abstract Background Expression of the HIV-1 vpr gene in human and fission yeast cells displays multiple highly conserved activities, which include induction of cell cycle G2 arrest and cell death. We have previously characterized a yeast heat shock protein 16 (Hsp16) that suppresses the Vpr activities when it is overproduced in fission yeast. Similar suppressive effects were observed when the fission yeast hsp16 gene was overexpressed in human cells or in the context of viral infection. In this study, we further characterized molecular actions underlying the suppressive effect of Hsp16 on the Vpr activities. Results We show that the suppressive effect of Hsp16 on Vpr-dependent viral replication in proliferating T-lymphocytes is mediated through its C-terminal end. In addition, we show that Hsp16 inhibits viral infection in macrophages in a dose-dependent manner. Mechanistically, Hsp16 suppresses Vpr activities in a way that resembles the cellular heat shock response. In particular, Hsp16 activation is mediated by a heat shock factor (Hsf)-dependent mechanism. Interestingly, vpr gene expression elicits a moderate increase of endogenous Hsp16 but prevents its elevation when cells are grown under heat shock conditions that normally stimulate Hsp16 production. Similar responsive to Vpr elevation of Hsp and counteraction of this elevation by Vpr were also observed in our parallel mammalian studies. Since Hsf-mediated elevation of small Hsps occurs in all eukaryotes, this finding suggests that the anti-Vpr activity of Hsps is a conserved feature of these proteins. Conclusion These data suggest that fission yeast could be used as a model to further delineate the potential dynamic and antagonistic interactions between HIV-1 Vpr and cellular heat shock responses involving Hsps.