Galactosaminogalactan and Pel are cationic heteropolysaccharides produced by the opportunistic pathogens Aspergillus fumigatus and Pseudomonas aeruginosa, respectively. These exopolysaccharides both contain 1,4-linked N-acetyl-D-galactosamine and play an important role in biofilm formation by these organisms. Proteins containing glycoside hydrolase domains have recently been identified within the biosynthetic pathway of each exopolysaccharide. Recombinant hydrolase domains from these proteins (Sph3 h from A. fumigatus and PelA h from P. aeruginosa) were found to degrade their respective polysaccharides in vitro. We therefore hypothesized that these glycoside hydrolases could exhibit antibiofilm activity and, further, given the chemical similarity between galactosaminogalactan and Pel, that they might display cross-species activity. Treatment of A. fumigatus with Sph3 h disrupted A. fumigatus biofilms with an EC 50 of 0.4 nM. PelA h treatment also disrupted preformed A. fumigatus biofilms with EC 50 values similar to those obtained for Sph3 h . In contrast, Sph3 h was unable to disrupt P. aeruginosa Pel-based biofilms, despite being able to bind to the exopolysaccharide. Treatment of A. fumigatus hyphae with either Sph3 h or PelA h significantly enhanced the activity of the antifungals posaconazole, amphotericin B, and caspofungin, likely through increasing antifungal penetration of hyphae. Both enzymes were noncytotoxic and protected A549 pulmonary epithelial cells from A. fumigatus-induced cell damage for up to 24 h. Intratracheal administration of Sph3 h was well tolerated and reduced pulmonary fungal burden in a neutropenic mouse model of invasive aspergillosis. These findings suggest that glycoside hydrolases can exhibit activity against diverse microorganisms and may be useful as therapeutic agents by degrading biofilms and attenuating virulence.biofilm | Aspergillus | Pseudomonas | therapeutics | exopolysaccharide
Mammalian cell culture has been used in many biological studies on the assumption that a cell line comprises putatively homogeneous clonal cells, thereby sharing similar phenotypic features. This fundamental assumption has not yet been fully tested; therefore, we developed a method for the chronological analysis of individual HeLa cells. The analysis was performed by live cell imaging, tracking of every single cell recorded on imaging videos, and determining the fates of individual cells. We found that cell fate varied significantly, indicating that, in contrast to the assumption, the HeLa cell line is composed of highly heterogeneous cells. Furthermore, our results reveal that only a limited number of cells are immortal and renew themselves, giving rise to the remaining cells. These cells have reduced reproductive ability, creating a functionally heterogeneous cell population. Hence, the HeLa cell line is maintained by the limited number of immortal cells, which could be putative cancer stem cells.
The affinity of integrin-ligand interaction is regulated extracellularly by divalent cations and intracellularly by inside-out signaling. We report here that the extracellular, membrane-proximal ␣/ stalk interactions not only regulate cation-induced integrin activation but also play critical roles in propagating inside-out signaling. Two closely related integrins, ␣IIb3 and ␣V3, share high structural homology and bind to similar ligands in an RGD-dependent manner. Despite these structural and functional similarities, they exhibit distinct responses to Mn 2؉ . Although ␣V3 showed robust ligand binding in the presence of Mn 2؉ , ␣IIb3 showed a limited increase but failed to achieve full activation. Swapping ␣ stalk regions between ␣IIb and ␣V revealed that the ␣ stalk, but not the ligand-binding head region, was responsible for the difference. A series of ␣IIb/␣V domain-swapping chimeras were constructed to identify the responsible domain. Surprisingly, the minimum component required to render ␣IIb3 susceptible to Mn 2؉ activation was the ␣V calf-2 domain, which does not contain any divalent cation-binding sites. The calf-2 domain makes interface with  epidermal growth factor 4 and  tail domain in three-dimensional structure. The effect of calf-2 domain swapping was partially reproduced by mutating the specific amino acid residues in the calf-2/epidermal growth factor 4- tail domain interface. When this interface was constrained by an artificially introduced disulfide bridge, the Mn 2؉ -induced ␣V3-fibrinogen interaction was significantly impaired. Notably, a similar disulfide bridge completely abrogated fibrinogen binding to ␣IIb3 when ␣IIb3 was activated by cytoplasmic tail truncation to mimic inside-out signaling. Thus, disruption/formation of the membraneproximal ␣/ stalk interface may act as an on/off switch that triggers integrin-mediated bidirectional signaling.Integrins are a family of ␣/ heterodimeric transmembrane cell surface receptors that mediate cell-extracellular matrix and cell-cell interactions. The hallmark of integrin-dependent adhesive interaction is its regulation by intracellular signaling events (inside-out signaling) and by divalent cations. In addition to mediating adhesive interactions, liganded integrins initiate signals inside the cell to modify cell behavior (outside-in signaling) and thus play fundamental roles in numerous biological processes such as differentiation, cell survival, apoptosis, and cell motility (1). Integrin-mediated bidirectional signaling is accompanied by conformational change of the integrin structure. The crystal structure of ␣V3 extracellular domains revealed an unexpected bent conformer distinct from the extended conformer observed under electron microscope (2, 3). High-resolution electron microscopic observation on truncated recombinant ␣V3 has confirmed the presence of both conformers, suggesting that the transition from one conformer to another might take place under physiological conditions (4). However, integrin extension per se is not requ...
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