Reconstitution of bacteriorhodopsin into cyclic lipid vesicles

Bacteriorhodopsin (bR), a membrane protein that can generate a light-driven proton pump, was successfully reconstituted into vesicles composed of an artificial cyclic lipid that mimics archaeal membrane lipids. Unlike reconstituted bR in 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles, the net topology and structure of bR molecules in cyclic lipid vesicles are identical to those in the native purple membrane of Halobacterium salinarum.     

Alterations in manganese, copper, and zinc contents, and intracellular status of the metal-containing superoxide dismutase in human mesothelioma cells

BACKGROUND AND AIM: Molecular diagnostics and therapeutics of human mesothelioma using disease-related markers present major challenges in clinical practice. To identify biochemical alternations that would be markers of human mesothelioma, we measured the intracellular steady-state levels of biologically important trace metals such as manganese (Mn), copper (Cu), and zinc (Zn) in a human mesothelial cell line, MeT-5A, and in five human mesothelioma cell lines (MSTO-211H, NCI-H226, NCI-H2052, NCI-H2452, ACC-MESO-1) by inductively coupled plasma-mass spectrometry (ICP-MS). We also aimed to investigate whether the alterations were related to the intracellular status of metal-containing superoxide dismutase (SOD). RESULTS: There were no significant differences in the contents of the trace metals among MeT-5A, MSTO-211H, and ACC-MESO-1 cells. However, each of the other three mesothelioma cell lines had a unique characteristic in terms of the intracellular amounts of the metals; NCI-H226 contained an extremely high level of Mn, an amount 7.3-fold higher than that in MeT-5A. NCI-H2052 had significantly higher amounts of Cu (3.4-fold) and Zn (1.3-fold) compared with MeT-5A. NCI-H2452 contained about 5.8-fold the amount of Cu and 2.5-fold that of Mn compared with MeT-5A. As for the intracellular levels of copper/zinc-SOD (Cu/Zn-SOD) and manganese-SOD (Mn-SOD), those of Cu/Zn-SOD were relatively unchanged among the cells tested, and no notable correlation with Cu or Zn contents was observed. On the other hand, all mesothelioma cells highly expressed Mn-SOD compared with MeT-5A, and a very high expression of the enzyme with a robust activity was observed in the two mesothelioma cells (NCI-H226, NCI-H2452) containing a large amount of Mn. CONCLUSIONS: In comparison with MeT-5A human mesothelial cells, some human mesothelioma cells had significantly higher amounts of Mn or Cu and one mesothelioma cell had a significantly higher amount of Zn. Interestingly, all mesothelioma cells overexpressed Mn-SOD compared with MeT-5A, and the cells whose Mn-SOD activity was increased contained higher amounts of Mn. It seemed that intracellular Mn content was positively correlated with Mn-SOD, suggesting that the intracellular Mn level is associated with Mn-SOD activity. These biochemical signatures could be potential disease-related markers of mesothelioma.     

Drosophila optic lobe neuroblasts triggered by a wave of proneural gene expression that is negatively regulated by JAK/STAT

Neural stem cells called neuroblasts (NBs) generate a variety of neuronal and glial cells in the central nervous system of the Drosophila embryo. These NBs, few in number, are selected from a field of neuroepithelial (NE) cells. In the optic lobe of the third instar larva, all NE cells of the outer optic anlage (OOA) develop into either NBs that generate the medulla neurons or lamina neuron precursors of the adult visual system. The number of lamina and medulla neurons must be precisely regulated because photoreceptor neurons project their axons directly to corresponding lamina or medulla neurons. Here, we show that expression of the proneural protein Lethal of scute [L(1)sc] signals the transition of NE cells to NBs in the OOA. L(1)sc expression is transient, progressing in a synchronized and ordered ;proneural wave' that sweeps toward more lateral NEs. l(1)sc expression is sufficient to induce NBs and is necessary for timely onset of NB differentiation. Thus, proneural wave precedes and induces transition of NE cells to NBs. Unpaired (Upd), the ligand for the JAK/STAT signaling pathway, is expressed in the most lateral NE cells. JAK/STAT signaling negatively regulates proneural wave progression and controls the number of NBs in the optic lobe. Our findings suggest that NBs might be balanced with the number of lamina neurons by JAK/STAT regulation of proneural wave progression, thereby providing the developmental basis for the formation of a precise topographic map in the visual center.     

Structural insights shed light onto septin assemblies and function

While the original septin mutants were identified more than 30 years ago for their role in cytokinesis [Hartwell, LH: Genetic control of the cell division cycle in yeast. IV. Genes controlling bud emergence and cytokinesis. Exp Cell Res 1971, 69: 265-276], the architecture of septin complexes and higher order structures has remained a mystery up until very recently. Over the last few months a number of converging approaches have suddenly provided a wealth of structural information about the different levels of septin organization. Here, we review these advancements and highlight their functional consequences.     

Allicin inhibits cell growth and induces apoptosis through the mitochondrial pathway in HL60 and U937 cells

In this article, the effects of allicin, a biological active compound of garlic, on HL60 and U937 cell lines were examined. Allicin induced growth inhibition and elicited apoptotic events such as blebbing, mitochondrial membrane depolarization, cytochrome c release into the cytosol, activation of caspase 9 and caspase 3 and DNA fragmentation. Pretreatment of HL60 cells with cyclosporine A, an inhibitor of the mitochondrial permeability transition pore (mPTP), inhibited allicin-treated cell death. HL60 cell survival after 1 h pretreatment with cyclosporine A, followed by 16 h in presence of allicin (5 microM) was approximately 80% compared to allicin treatment alone (approximately 50%). Also N-acetyl cysteine, a reduced glutathione (GSH) precursor, prevented cell death. The effects of cyclosporine A and N-acetyl cysteine suggest the involvement of mPTP and intracellular GSH level in the cytotoxicity. Indeed, allicin depleted GSH in the cytosol and mitochondria, and buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly augmented allicin-induced apoptosis. In HL60 cells treated with allicin (5 microM, 30 min) the redox state for 2GSH/oxidized glutathione shifted from EGSH -240 to -170 mV. The same shift was observed in U937 cells treated with allicin at a higher concentration for a longer period of incubation (20 microM, 2 h). The apoptotic events induced by various concentrations of allicin correlate to intracellular GSH levels in the two cell types tested (HL60: 3.7 nmol/10(6) cells; U937: 7.7 nmol/10(6) cells). The emerging mechanistic basis for the antiproliferative function of allicin, therefore, involves the activation of the mitochondrial apoptotic pathway by GSH depletion and by changes in the intracellular redox status.     

An FGF1:FGF2 chimeric growth factor exhibits universal FGF receptor specificity, enhanced stability and augmented activity useful for epithelial proliferation and radioprotection

Structural instability of wild-type fibroblast growth factor (FGF)-1 and its dependence on exogenous heparin for optimal activity diminishes its potential utility as a therapeutic agent. Here we evaluated FGFC, an FGF1:FGF2 chimeric protein, for its receptor affinity, absolute heparin-dependence, stability and potential clinical applicability. Using BaF3 transfectants overexpressing each FGF receptor (FGFR) subtype, we found that, like FGF1, FGFC activates all of the FGFR subtypes (i.e., FGFR1c, FGFR1b, FGFR2c, FGFR2b, FGFR3c, FGFR3b and FGFR4) in the presence of heparin. Moreover, FGFC activates FGFRs even in the absence of heparin. FGFC stimulated keratinocytes proliferation much more strongly than FGF2, as would be expected from its ability to activate FGFR2b. FGFC showed greater structural stability, biological activity and resistance to trypsinization, and less loss in solution than FGF1 or FGF2. When FGFC was intraperitoneally administered to BALB/c mice prior to whole body gamma-irradiation, survival of small intestine crypts was significantly enhanced, as compared to control mice. These results suggest that FGFC could be useful in a variety of clinical applications, including promotion of wound healing and protection against radiation-induced damage.     

Analyses of functional interaction between RECQL1, RECQL5, and BLM which physically interact with DNA topoisomerase IIIalpha

RECQL1 and RECQL5 as well as BLM reportedly interact with TOP3alpha whose defect is lethal for the cell. Therefore in this study, we characterized recql5/recql1/blm triple mutants from DT40 cells to determine whether the triple mutants show a top3alpha disrupted cell-like phenotype. The triple mutants are viable. Moreover, both blm/recql1 and recql5/blm cells, and recql5/recql1/blm cells grew slightly slower than blm cells, that is, triple mutant cells grew almost the same rate as either of the double mutant cells. The blm cells showed sensitivity to methyl methanesulfonate (MMS) and ultraviolet light (UV), about a 10-fold increase in sister chromatid exchange (SCE), and about a 3-fold increase in damage-induced mitotic chiasma compared to wild-type cells. The triple mutants showed the same sensitivity to MMS or UV and the same frequency of damage-induced mitotic chiasma compared to those of blm cells, indicating that unlike BLM, RECQL1 and RECQL5 play a little role in the repair of or tolerance to DNA damages. However, recql5/blm cells showed higher frequency of SCE than blm cells, whereas the RECQL1 gene disruption had no effect on SCE in blm cells and even in recql5/blm cells.     

Sequence variation of Vanabin2-like vanadium-binding proteins in blood cells of the vanadium-accumulating ascidian Ascidia sydneiensis samea

The blood cells of ascidians accumulate extremely high levels of the transition metal vanadium. We previously isolated four vanadium-binding proteins (Vanabins 1-4) and a homologous protein (VanabinP) from the vanadium-rich ascidian Ascidia sydneiensis samea. In the present study, we identified cDNAs encoding five different Vanabin2-related proteins in A. sydneiensis samea blood cells. It was notable that the sequences of the encoded proteins vary from that of Vanabin2 at up to 14 specific positions, while both the polypeptide length and the 18 cysteine residues were completely conserved. The most divergent protein, named 14MT, differed from Vanabin2 at all 14 positions. Using immobilized metal-ion affinity chromatography, we found that Vanabin2 and 14MT have the same metal-ion selectivity, but the overall affinity of 14MT for VO(2+) is higher than that of Vanabin2. Binding number for VO(2+) ions was the same between Vanabin2 and 14MT as assessed by gel filtration. These results suggested that sequence variations were under strict evolutionary constraints and high-affinity binding sites for VO(2+) are conserved among Vanabin2 variants.