Membrane cholesterol depletion enhances ligand binding function of human serotonin1A receptors in neuronal cells

Membrane lipid composition of cells in the nervous system is unique and displays remarkable diversity. Cholesterol metabolism and homeostasis in the central nervous system and their role in neuronal function represent important determinants in neuropathogenesis. The serotonin_1A receptor is an important member of the G-protein coupled receptor superfamily, and is involved in a variety of cognitive, behavioral, and developmental functions. We report here, for the first time, that the ligand binding function of human serotonin_1A receptors exhibits an increase in membranes isolated from cholesterol-depleted neuronal cells. Our results gain pharmacological significance in view of the recently described structural evidence of specific cholesterol binding site(s) in GPCRs, and could be useful in designing better therapeutic strategies for neurodegenerative diseases associated with GPCRs.     

CD90 and CD110 correlate with cancer stem cell potentials in human T-acute lymphoblastic leukemia cells

Although cancer stem cells (CSCs) have been recently identified in myeloid leukemia, published data on lymphoid malignancy have been sparse. T-acute lymphoblastic leukemia (T-ALL) is characterized by the abnormal proliferation of T-cell precursors and is generally aggressive. As CD34 is the only positive-selection marker for CSCs in T-ALL, we performed extensive analysis of CD markers in T-ALL cell lines. We found that some of the tested lines consisted of heterogeneous populations of cells with various levels of surface marker expression. In particular, a small subpopulation of CD90 (Thy-1) and CD110 (c-Mpl) were shown to correlate with stem cell properties both in vitro and in transplantation experiments. As these markers are expressed on hematopoietic stem cells, our results suggest that stem cell-like population are enriched in CD90+/CD110+ fraction and they are useful positive-selection markers for the isolation of CSCs in some cases of T-ALL.     

A continuous microplate assay for sirtuins and nicotinamide-producing enzymes

Nicotinamide adenine dinucleotide (NAD⁺)-dependent protein deacetylases (sirtuins) and other enzymes that produce nicotinamide are integral to many cellular processes. Yet current activity measurements involve expensive and time-consuming assays. Here we present a spectroscopic assay that circumvents many issues of previous methods. This assay permits continuous product monitoring over time, allows determination of steady-state kinetic parameters, and is readily adaptable to high-throughput screening. The methodology uses an enzyme-coupled system in which nicotinamide is converted to nicotinic acid and ammonia by nicotinamidase. The ammonia is transferred to α-ketoglutarate via glutamate dehydrogenase, yielding glutamate and the oxidation of NAD(P)H to NAD(P)⁺, which is measured spectrophotometrically at 340 nm. Using this continuous assay with sirtuin-1 (Sirt1) and the ADP-ribosyl cyclase CD38, the resulting steady-state kinetic parameters are in excellent agreement with values obtained by other published methods. Importantly, this assay permitted determination of k_cat and K_m values with the native acetylated substrate acetyl-CoA synthetase-1; measurement of Sirt1, Sirt2, and Sirt3 activities from mammalian cell extracts; and determination of IC₅₀ values of various Sirt1 inhibitors. This assay is applicable to any nicotinamide-forming enzyme and will be an important tool to address many outstanding questions surrounding their regulation.     

Binding of amphipathic α-helical antimicrobial peptides to lipid membranes: Lessons from temporins B and L

Temporins constitute a family of amphipathic α-helical antimicrobial peptides (AMP) and contain some of the shortest cytotoxic peptides, comprised of only 10-14 residues. General characteristics of temporins parallel those of other AMP, both in terms of structural features and biophysical properties relating to their interactions with membrane lipids, with selective lipid-binding properties believed to underlie the discrimination between target vs host cells. Lipid-binding properties also contribute to the cytotoxicity AMP, causing permeabilization of their target cell membranes. The latter functional property of AMP involves highly interdependent acidic phospholipid-induced conformational changes, aggregation, and formation of toxic oligomers in the membrane. These oligomers are subsequently converted to amyloid-type fibers, as demonstrated for e.g. temporins B and L in our laboratory, and more recently for dermaseptins by Auvynet et al. Amyloid state represents the generic minimum in the folding/aggregation free energy landscape, and for AMP its formation most likely serves to detoxify the peptides, in keeping with the current consensus on mature amyloid being inert and non-toxic. The above scenario is supported by sequence analyses of temporins as well as other amphipathic α-helical AMP belonging to diverse families. Accordingly, sequence comparison identifies ‘conformational switches’, domains with equal probabilities for adopting random coil, α-helical and β-sheet structures. These regions were further predicted also to aggregate and assemble into amyloid β-sheets. Taken together, the lipid-binding properties and structural characterization lend support to the notion that the mechanism of membrane permeabilization by temporins B and L and perhaps of most AMP could be very similar, if not identical, to that of the paradigm amyloid forming cytotoxic peptides, responsible for degenerative cell loss in e.g. prion, Alzheimer's and Parkinson's disease, and type 2 diabetes.     

Loss of membrane cholesterol influences lysosomal permeability to potassium ions and protons

Cholesterol is an essential component of lysosomal membranes. In this study, we investigated the effects of membrane cholesterol on the permeability of rat liver lysosomes to K⁺ and H⁺, and the organelle stability. Through the measurements of lysosomal β-hexosaminidase free activity, membrane potential, membrane fluidity, intra-lysosomal pH, and lysosomal proton leakage, we established that methyl-β-cyclodextrin (MβCD)-produced loss of membrane cholesterol could increase the lysosomal permeability to both potassium ions and protons, and fluidize the lysosomal membranes. As a result, potassium ions entered the lysosomes through K⁺/H⁺ exchange, which produced osmotic imbalance across the membranes and osmotically destabilized the lysosomes. In addition, treatment of the lysosomes with MβCD caused leakage of the lysosomal protons and raised the intra-lysosomal pH. The results indicate that membrane cholesterol plays important roles in the maintenance of the lysosomal limited permeability to K⁺ and H⁺. Loss of this membrane sterol is critical for the organelle acidification and stability.     

Hematopoiesis-dependent expression of CD44 in murine hepatic progenitor cells

The fetal liver serves as the predominant hematopoietic organ until birth. However, the mechanisms underlying this link between hematopoiesis and hepatogenesis are unclear. Previously, we reported the isolation of a monoclonal antibody (anti-Liv8) that specifically recognizes an antigen (Liv8) present in murine fetal livers at embryonic day 11.5 (E11.5). Liv8 is a cell surface molecule expressed by hematopoietic cells in both fetal liver and adult mouse bone marrow. Here, we report that Liv8 is also transiently expressed by hepatoblasts at E11.5. Using protein purification and mass spectrometry, we have identified Liv8 as the CD44 protein. Interestingly, the expression of Liv8/CD44 in fetal liver was completely lost in AML1^−/− murine embryos, which lack definitive hematopoiesis. These results show that hepatoblasts change from Liv8/CD44-negative to Liv8/CD44-positive status in a hematopoiesis-dependent manner by E11.5, and indicate that Liv8/CD44 expression is an important link between hematopoiesis and hepatogenesis during fetal liver development.     

CD9 correlates with cancer stem cell potentials in human B-acute lymphoblastic leukemia cells

Cancer stem cell (CSC) theory suggests that only a small subpopulation of cells having stem cell-like potentials can initiate tumor development. While recent data on acute lymphoblastic leukemia (ALL) are conflicting, some studies have demonstrated the existence of such cells following CD34-targeted isolation of primary samples. Although CD34 is a useful marker for the isolation of CSCs in leukemias, the identification of other specific markers besides CD34 has been relatively unsuccessful. To identify new markers, we first performed extensive analysis of surface markers on several B-ALL cell lines. Our data demonstrated that every B-ALL cell line tested did not express CD34 but certain lines contained cell populations with marked heterogeneity in marker expression. Moreover, the CD9⁺ cell population possessed stem cell characteristics within the clone, as demonstrated by in vitro and transplantation experiments. These results suggest that CD9 is a useful positive-selection marker for the identification of CSCs in B-ALL.     

The role of CD4 T cell help for CD8 CTL activation

CD4 T cells play an important role in the initiation and persistence of CD8 T cells responses. In this review, we report on and evaluate the mechanisms by which CD4 T cells contribute to activation of CD8 T cells and the signal pathways of the down-streaming events after CD4 T cell help.     

Nectin-3 expression is elevated in limbal epithelial side population cells with strongly expressed stem cell markers

Corneal epithelial stem cells (CESCs) are essential for maintaining the ocular surface. However, the lack of surface markers for CESCs remains a serious obstacle in the identification of CESCs. Previously, we showed that rabbit limbal epithelial side population (rLE-SP) cells exhibited stem cell phenotypes including increased expression of CD61, a marker for mouse hematopoietic stem cells. Here, we demonstrate that nectin-3, an immunoglobulin-like cell-cell adhesion molecule, is highly expressed in rLE-SP cells. Additionally, nectin-3⁺ cells were significantly enriched among CD61⁺rLE-SP cells as compared to CD61⁻rLE-SP cells. In mouse bone marrow side population cells, a correlation between expression of nectin-3 and CD61 was also observed. These data strongly suggest that nectin-3 may contribute to the identification of CESCs.