Intracellular Proton Access in a Cl?/H+ Antiporter

Chloride-transporting membrane proteins of the CLC family appear in two distinct mechanistic flavors: H⁺-gated Cl⁻ channels and Cl⁻/H⁺ antiporters. Transmembrane H⁺ movement is an essential feature of both types of CLC. X-ray crystal structures of CLC antiporters show the Cl⁻ ion pathway through these proteins, but the H⁺ pathway is known only inferentially by two conserved glutamate residues that act as way-stations for H⁺ in its path through the protein. The extracellular-facing H⁺ transfer glutamate becomes directly exposed to aqueous solution during the transport cycle, but the intracellular glutamate E203, Glu_in, is buried within the protein. Two regions, denoted “polar” and “interfacial,” at the intracellular surface of the bacterial antiporter CLC-ec1 are examined here as possible pathways by which intracellular aqueous protons gain access to Glu_in. Mutations at multiple residues of the polar region have little effect on antiport rates. In contrast, mutation of E202, a conserved glutamate at the protein–water boundary of the interfacial region, leads to severe slowing of the Cl⁻/H⁺ antiport rate. An X-ray crystal structure of E202Y, the most strongly inhibited of these substitutions, shows an aqueous portal leading to Glu_in physically blocked by cross-subunit interactions; moreover, this mutation has only minimal effect on a monomeric CLC variant, which necessarily lacks such interactions. The several lines of experiments presented argue that E202 acts as a water-organizer that creates a proton conduit connecting intracellular solvent with Glu_in.     

Multiparametric assessment of Cd2? cytotoxicity using MTT-based microfluidic image cytometry

A modified MTT protocol-based microfluidic image cytometry (μFIC) was performed to assess Cd(2+) induced cytotoxicity. The expanded capabilities of μFIC, such as in situ measurement, high-throughput, and multiparametric analysis of adherent cells under precisely controlled chemical environments of microfluidic channels, were demonstrated in this study. Multiparametric analysis of μFIC data has enabled us to categorize the progress of cell death into at least four different subgroups based on their morphology and metabolic activity. These advantages of the MTT-based μFIC as a simpler, cheaper, and faster in vitro cell-based assay tool have many implications in biomedical, pharmaceutical, toxicological, and biological application areas, and we propose this technique as a future high throughput-high content screening (HT-HCS) platform for cytotoxicity assays and drug screening.     

Chemokine Receptor Ccr1 Drives Neutrophil-Mediated Kidney Immunopathology and Mortality in Invasive Candidiasis

Invasive candidiasis is the 4^th leading cause of nosocomial bloodstream infection in the US with mortality that exceeds 40% despite administration of antifungal therapy; neutropenia is a major risk factor for poor outcome after invasive candidiasis. In a fatal mouse model of invasive candidiasis that mimics human bloodstream-derived invasive candidiasis, the most highly infected organ is the kidney and neutrophils are the major cellular mediators of host defense; however, factors regulating neutrophil recruitment have not been previously defined. Here we show that mice lacking chemokine receptor Ccr1, which is widely expressed on leukocytes, had selectively impaired accumulation of neutrophils in the kidney limited to the late phase of the time course of the model; surprisingly, this was associated with improved renal function and survival without affecting tissue fungal burden. Consistent with this, neutrophils from wild-type mice in blood and kidney switched from Ccr1^lo to Ccr1^high at late time-points post-infection, when Ccr1 ligands were produced at high levels in the kidney and were chemotactic for kidney neutrophils ex vivo. Further, when a 1∶1 mixture of Ccr1^+/+ and Ccr1^−/− donor neutrophils was adoptively transferred intravenously into Candida-infected Ccr1^+/+ recipient mice, neutrophil trafficking into the kidney was significantly skewed toward Ccr1^+/+ cells. Thus, neutrophil Ccr1 amplifies late renal immunopathology and increases mortality in invasive candidiasis by mediating excessive recruitment of neutrophils from the blood to the target organ.     

Identification of high affinity polo-like kinase 1 (Plk1) polo-box domain binding peptides using oxime-based diversification

In an effort to develop improved binding antagonists of the polo-like kinase 1 (Plk1) polo-box domain (PBD), we optimized interactions of the known high affinity 5-mer peptide PLHSpT using oxime-based post solid-phase peptide diversification of the N-terminal Pro residue. This allowed us to achieve up to two orders of magnitude potency enhancement. An X-ray crystal structure of the highest affinity analogue in complex with Plk1 PBD revealed new binding interactions in a hydrophobic channel that had been occluded in X-ray structures of the unliganded protein. This study represents an important example where amino acid modification by post solid-phase oxime ligation can facilitate the development of protein-protein interaction inhibitors by identifying new binding pockets that would not otherwise be accessible to coded amino acid residues.     

Preventive effects of protopanaxadiol and protopanaxatriol ginsenosides on liver inflammation and apoptosis in hyperlipidemic apoE KO mice

Ginsenosides, bioactive compounds of Panax GinsengC.A. Meyer, are divided into protopanaxadiol (PD) and protopanaxtriol (PT). The aim of this study was to evaluate the protective effects of different PD and PT combination ratios on liver inflammation and apoptosis in hyperlipidemic apo E KO mice. R1 (PD/PT = 1, high Rg₁ and Rb₁) and R2 (PD/PT = 2, high Re and Rd) extracts were intraperitoneally injected by 100 mg/kg/day at the 8th week. R1 and R2 improved atherogenic indices by increasing HDL and lowering total cholesterol (TC) and triacylglyceride (TG) selectively. R1 decreased lipid peroxides (LPO) level in plasma and liver tissue of hyperlipidemic mice, and R2 lowered plasma malondialdehyde(MDA) level. R1 and R2 not only regulated the expression of cyclooxygenase (COX)-2, IκB-α, phopho-ERK 1/2, and phopho-SAPK/JNK levels but also were significantly effective in blocking apoptotic signals, such as caspase-8, -9, as well as the cleavage of PARP in liver. Different combinational treatment of PD and PT extracts might ameliorate the liver inflammation and apoptosis in hyperlipidemic apo E KO mice, which is atherosclerotic animal model.     

Design and synthesis of 4-O-methylhonokiol analogs as inhibitors of cyclooxygenase-2 (COX-2) and PGF₁ production

A series of novel 4-O-methylhonokiol analogs were synthesized in light of revealing structure-activity relationship for inhibitory effect of COX-2 enzyme. The key strategy of the molecular design was oriented towards modification of the potential metabolic soft spots (e.g., phenol and olefin) or by altering the polar surface area via incorporating heterocycles such as isoxazole and triazole. Most of all exhibited the inhibitory effects on COX-2 and PGF(1) production but not macrophage NO production. Especially, aryl carbamates 10 and 11 exhibited more potent inhibitory activity against COX-2 and PGF(1) production.     

Leveraging kinase inhibitors to develop small molecule tools for imaging kinases by fluorescence microscopy

As the usage of fluorescence microscopy as a tool to study biological systems continues to grow, so does the need for additional tools that permit the selective detection of proteins of interest. Existing selective and well-characterized kinase inhibitors may be exploited to develop novel small molecule probes useful in imaging kinases by fluorescence microscopy.     

N-terminal cleavage fragment of focal adhesion kinase is required to activate the survival signalling pathway in cultured myoblasts under oxidative stress

We have previously shown that the cultured L6 myoblasts are susceptible to menadione-induced oxidative stress. Damaged cells were detached from the culture dishes. In the present study, we focused on focal adhesion kinase (FAK), which plays pivotal roles in maintaining focal adhesion function and cell survival. FAK, normally localized at the focal adhesion regions of the myoblasts, was not observed at the regions under oxidative stress induced by menadione and H(2) O(2) . Two cleavage products, 80-kDa N-terminal FAK and 35-kDa C-terminal FAK fragments, as well as full-length FAK (125?kDa) were detected in myoblasts cultured under normal conditions by western blotting with anti-N-terminal FAK or anti-C-terminal FAK sera. Of interest was the finding that the cleavage products of FAK (but not full-length FAK) disappeared under oxidative stress. The cleavage of full-length FAK to N-terminal FAK and C-terminal FAK was inhibited by calpeptin, a specific calpain inhibitor. In addition, pre-incubation of cells with calpeptin resulted in a sharp decrease in survival signals, such as Akt phosphorylation and the ratio of Bcl-2/Bax, under stress conditions. By contrast, not only relative viability, but also Akt phosphorylation and the ratio of Bcl-2/Bax was significantly improved when cells were transfected with a DNA construct of N-terminal FAK-Myc. These results suggest that the N-terminal FAK positively regulates survival signalling in early phases of oxidative stress in the cultured myoblasts.     

Cold-induced activation of brown adipose tissue and adipose angiogenesis in mice

Exposure of humans and rodents to cold activates thermogenic activity in brown adipose tissue (BAT). This protocol describes a mouse model to study the activation of BAT and angiogenesis in adipose tissues by cold acclimation. After a 1-week exposure to 4 °C, adult C57BL/6 mice show an obvious transition from subcutaneous white adipose tissue (WAT) into brown-like adipose tissue (BRITE). The BRITE phenotype persists after continuous cold exposure, and by the end of week 5 BRITE contains a high number of uncoupling protein-1-positive mitochondria, a characteristic feature of BAT. During the transition from WAT into BRITE, the vascular density is markedly increased owing to the activation of angiogenesis. In BAT, cold exposure stimulates thermogenesis by increasing the mitochondrial content and metabolic rate. BAT and the increased metabolic rate result in a lean phenotype. This protocol provides an outstanding opportunity to study the molecular mechanisms that control adipose mass.     

Revised classification/nomenclature of vitiligo and related issues: the Vitiligo Global Issues Consensus Conference

During the 2011 International Pigment Cell Conference (IPCC), the Vitiligo European Taskforce (VETF) convened a consensus conference on issues of global importance for vitiligo clinical research. As suggested by an international panel of experts, the conference focused on four topics: classification and nomenclature; definition of stable disease; definition of Koebner's phenomenon (KP); and 'autoimmune vitiligo'. These topics were discussed in seven working groups representing different geographical regions. A consensus emerged that segmental vitiligo be classified separately from all other forms of vitiligo and that the term 'vitiligo' be used as an umbrella term for all non-segmental forms of vitiligo, including 'mixed vitiligo' in which segmental and non-segmental vitiligo are combined and which is considered a subgroup of vitiligo. Further, the conference recommends that disease stability be best assessed based on the stability of individual lesions rather than the overall stability of the disease as the latter is difficult to define precisely and reliably. The conference also endorsed the classification of KP for vitiligo as proposed by the VETF (history based, clinical observation based, or experimentally induced). Lastly, the conference agreed that 'autoimmune vitiligo' should not be used as a separate classification as published evidence indicates that the pathophysiology of all forms of vitiligo likely involves autoimmune or inflammatory mechanisms.