Chloride in airway smooth muscle: the ignored anion no longer?

This Perspectives accompanies an Editorial Focus that summarizes new developments concerning the role of chloride in airway smooth muscle physiology. We provide several observations and mechanistic insights to reconcile recent experimental evidence with existing paradigms concerning chloride channel-mediated effects on airway smooth muscle tone. In addition, we highlight the potentially complex and dynamic nature that chloride currents and membrane potential have on calcium handling and airway smooth muscle contractility.     

Use of a library of mutated Maackia amurensis hemagglutinin for profiling the cell lineage and differentiation

Thirty-five variant lectins were prepared by mutations of two amino acids within the carbohydrate-recognition domain of Maackia amurensis hemagglutinin (MAH). Each lectin showed unique carbohydrate specificity according to their bindings to soluble polyacrylamide with various mono- and oligosaccharides and to glycophorin A. The relative intensity of the bindings of carcinoma, myeloid, fibroblastic, and melanoma cells to immobilized MAH variant lectins was examined. Each cell line showed distinct profiles regarding the number of cells bound to wild-type and 35 MAH variants and the differences and the similarities in these binding profiles were quantitatively documented by the cluster analysis. The cell lines were classified into several groups and these groups surprisingly corresponded to the lineage of the cells. These results indicated that a library of mutated MAH is useful as a tool for the profiling of various cells based on the variations of the surface glycans.     

PLK1/vimentin signaling facilitates immune escape by recruiting Smad2/3 to PD-L1 promoter in metastatic lung adenocarcinoma

The prerequisite function of vimentin for the epithelial–mesenchymal transition (EMT) is not clearly elucidated yet. Here, we show that vimentin phosphorylated by PLK1, triggers TGF-β-signaling, which consequently leads to metastasis and PD-L1 expression for immune suppression in lung adenocarcinoma. The clinical correlation between expression of both vimentin and PLK1, and overall survival rates of patients was significant in lung adenocarcinoma but not in squamous cell carcinoma. The phosphorylation of vimentin was accompanied by the activation of PLK1 during TGF-β-induced EMT in lung adenocarcinoma. Among the several phosphorylation sites determined by phospho-proteomic analysis and the site-specific mutagenesis, the phosphorylation at S339 displayed the most effective metastasis and tumourigenesis with the highest expression of PD-L1, compared with that of wild-type and other versions in both 3D cell culture and tail-vein injection metastasis models. Phosphomimetic vimentin at S339 interacted with p-Smad2 for its nuclear localization, leading to the expression of PD-L1. Clinical relevance revealed the inverse correlation between the survival rates of patients and the expressions of VIM, PLK1, and CD274 in primary and metastatic lung adenocarcinoma. Thus, PLK1-mediated phosphorylation of vimentin activates TGF-β signaling pathway, leading to the metastasis and immune escape through the expression of PD-L1, functioning as a shuttling protein in lung adenocarcinoma.Subject terms: Cancer microenvironment, Prognostic markers     

Femcoat, a novel eggshell protein in Drosophila: functional analysis by double stranded RNA interference.

In Drosophila oogenesis, follicle cells derived from somatic tissue surround the oocyte and play key roles in generating properly polarized oocytes. During the later steps of oogenesis, follicle cells are involved in secretion of proteins that make the eggshell, an essential protective layer for the oocyte. Although studies on the signaling processes to make polarized oocytes have been progressed very far, studies on the mechanisms for eggshell formation is not clear yet. To elucidate the underlying mechanism in eggshell formation, we used a differential display screen to isolate genes that are specifically expressed during the later stages of oogenesis, and isolated a novel gene, Femcoat. Femcoat encodes a putative chorion membrane protein that contains many highly charged residues and has a putative signal peptide. Femcoat is expressed specifically in the follicle cells with a punctate staining pattern typical of secreted proteins, and becomes cross-linked heavily at the final steps of oogenesis. To identify the developmental role of Femcoat in eggshell formation, we performed an inducible double stranded RNA mediated interference (dsRNAi) method to specifically reduce Femcoat expression during oogenesis in adult flies. Electron microscopy analysis of egg chambers from these flies showed defects in chorion formation. These pieces of evidence demonstrated that Femcoat is necessary for eggshell formation, especially during chorion synthesis. Our results demonstrate that inducible dsRNAi analysis can be effective in determining the developmental function of novel genes.     

High-level secretory production of human granulocyte-colony stimulating factor by fed-batch culture of recombinant Escherichia coli

Secretory production of human granulocyte colony-stimulating factor fusion protein (hG-CSF) by fed-batch culture of Escherichia coli was investigated in both 2.5-L and 30-L fermentors. To develop a fed-batch culture condition that allows efficient production of hG-CSF, different feeding strategies including pH-stat, exponential and constant feeding were examined. Among these, the constant feeding strategy (0.228 g glucose2min^-1) and the exponential feeding that supports a low specific growth rate (µ=0.116 h^-1) resulted in the best hG-CSF production. Under these conditions, 4.4 g2L^-1 of hG-CSF was produced. The effect of induction time on the protein production was also investigated. For the fed-batch cultures carried out with the pH-stat and exponential feeding strategies, induction at higher cell density (late-exponential phase) resulted in more hG-CSF production compared with induction at lower cell density (early to mid-exponential phase). The constant feeding strategy that supported best hG-CSF production was applied to the scale-up production of hG-CSF in 30 L of fermentor. The maximum dry cell weight and hG-CSF concentration of 51.7 and 4.2 g2L^-1, respectively, was obtained.     

Opioids, feeding, and anorexias

This review summarizes recent work that focuses on the role of endogenous opioids (EOs) and opiate receptors in the control of food intake. Although the anorexic effect of opiate antagonists are now well accepted, the exact EO, site(s), and mechanism(s) of action remain to be established. However, accumulating evidence suggests that dynorphin, an endogenous ligand for kappa-type opiate receptors, is an important regulator (stimulant) of appetite. The roles of other EOs, such as beta-endorphin, are less clear. EOs appear to be involved in maintaining normal feeding behavior and are likely responsible for the overconsumption of fat in genetically obese and stressed subjects. Opiate antagonists block overconsumption of palatable foods, thus offering a promising approach to weight reduction for some overweight individuals. Anorexias may follow from a deficiency of kappa-type opioid activity, and surprisingly, can also result from excess opioid activity. Indeed, opiate antagonists of the mu type (naloxone) can enhance eating and weight gain in certain anorexic conditions. Therefore, it appears that excess opioid agonist activity may result in hyperphagia or anorexia (depending on the opiate receptor type). Finally, opiate antagonists may help normalize both types of pathological feeding states.