Spatiotemporal calcium dynamics in single astrocytes and its modulation by neuronal activity

Astrocytes produce a complex repertoire of Ca²⁺ events that coordinate their major functions. The principle of Ca²⁺ events integration in astrocytes, however, is unknown. Here we analyze whole Ca²⁺ events, which were defined as spatiotemporally interconnected transient Ca²⁺ increases. Using such analysis in single hippocampal astrocytes in culture and in slices we found that spreads and durations of Ca²⁺ events follow power law distributions, a fingerprint of scale-free systems. A mathematical model demonstrated that such Ca²⁺ dynamics can arise from intracellular inositol-3-phosphate diffusion. The power law exponent (α) was decreased by activation of metabotropic glutamate receptors (mGluRs) either by specific receptor agonist or by low frequency stimulation of glutamatergic fibers in hippocampal slices. Decrease in α indicated an increase in proportion of large Ca²⁺ events. Notably, mGluRs activation did not increase the frequency of whole Ca²⁺ events. This result suggests that neuronal activity does not trigger new Ca²⁺ events in astrocytes (detectable by our methods), but modulates the properties of existing ones. Thus, our results provide a new perspective on how astrocyte responds to neuronal activity by changing its Ca²⁺ dynamics, which might further affect local network by triggering release of gliotransmitters and by modulating local blood flow.     

Aralin,a type II ribosome-inactivating protein from Aralia elata, exhibits selective anticancer activity through the processed form of a 110-kDa high-density lipoprotein-binding protein: A promising anticancer drug

Aralin from Aralia elata is a newly identified type II ribosome- inactivating protein, which preferentially induces apoptosis in cancer cells. In this study, we identified that the aralin receptor is a 110-kDa high-density lipoprotein-binding protein (HDLBP), which functions as a HDL receptor. The sensitivities of tumor cell lines to aralin were dependent on the expression levels of the 110-kDa HDLBP and its forced expression in aralin-resistant Huh7 cells conferred aralin sensitivity. HDLBP-knockdown HeLa cells showed a significant aralin resistance in vitro and in vivo. Conversely, ectopic expression of the 150-kDa HDLBP resulted in increased aralin sensitivity in vivo, accompanying enhanced expression of the 110-kDa HDLBP. Thus, these results showed that the110-kDa HDLBP in lipid rafts acted as an aralin receptor and that its expression levels determined aralin sensitivity, suggesting that aralin could be a promising anticancer drug for HDLBP-overexpressing tumors.     

Increased expression of ERp57/GRP58 is protective against pancreatic beta cell death caused by autophagic failure

Autophagy is a tightly regulated self-digestion system. As in other cell types, autophagy plays an essential role in the homeostasis of pancreatic beta cells. However, the mechanisms involved in the deterioration of beta cell function caused by autophagic failure have not yet been fully elucidated. To gain insight into its mechanisms, we compared the protein expression of islets from beta cell-specific Atg7-deficient mice (Atg7^Δβ-cell mice) and their controls (Atg7^f/f mice). Liquid chromatography/mass spectrometry after 1-dimensional electrophoresis identified the increased expression of ERp57/GRP58 in islets isolated from Atg7^Δβ-cell mice compared with those from Atg7^f/f mice. The expression level of ERp57 was also elevated in rat insulinoma INS-1 cells by inducible knock-down of the atg7-gene. In Atg7 knock-down INS-1 cells, the suppression of ERp57 expression by siRNA resulted in an increase in the level of cleaved Caspase-3 protein and a decrease in the number of live cells. Furthermore, cell cycle analyses demonstrated that the suppressed expression of ERp57 increased the sub-G1 population. These data reveal that increased expression of ERp57 may contribute to the protection from beta cell death caused by autophagic failure.     

ADAMTS1 inhibits lymphangiogenesis by attenuating phosphorylation of the lymphatic endothelial cell-specific VEGF receptor

Angiogenesis and lymphangiogenesis play roles in malignant tumor progression, dissemination, and metastasis. ADAMTS1, a member of the matrix metalloproteinase family, is known to inhibit angiogenesis. Recombinant ADAMTS1 was shown to strongly inhibit angiogenesis. We investigated whether ADAMTS1 inhibited lymphangiogenesis in the present study. We examined cell proliferation and cell migration in normal human dermal lymphatic microvascular endothelial cells (HMVEC-dLy) transduced with or without adenoviral human ADAMTS1 gene therapy. We then examined the VEGFC/VEGFR3 signal transduction pathway in ADAMTS1-transduced HMVEC-dLy. Cell proliferation and tube formation in Matrigel were significantly lower with transduced ADAMTS1 than with control (non-transduced HMVEC-dLy). The phosphorylation of VEGFR3 was also attenuated by ADAMTS1 gene therapy in HMVEC-dLy. Immunoprecipitation assays revealed that ADAMTS1 formed a complex with VEGFC. Our results demonstrated that ADAMTS1 inhibited lymphangiogenesis in vitro. The data highlight the new function of ADAMTS1 in the regulation of lymphangiogenesis and the therapeutic potential of ADAMTS1 in cancer therapy.     

Purification,characterization, and cloning of the gene for a biodegradable plastic-degrading enzyme from Paraphoma-related fungal strain B47-9

Paraphoma-related fungal strain B47-9 secreted a biodegradable plastic (BP)-degrading enzyme which amounted to 68 % (w/w) of the total secreted proteins in a culture medium containing emulsified poly(butylene succinate-co-adipate) (PBSA) as sole carbon source. The gene for this enzyme was found to be composed of an open reading frame consisting of 681 nucleotides encoding 227 amino acids and two introns. Southern blot analysis showed that this gene exists as a single copy. The deduced amino acid sequence suggested that this enzyme belongs to the cutinase (E.C.3.1.1.74) family; thus, it was named P araphoma-related fungus cutinase-like enzyme (PCLE). It degraded various types of BP films, such as poly(butylene succinate), PBSA, poly(butylene adipate-co-terephthalate), poly(ε-caprolactone), and poly(dl-lactic acid). It has a molecular mass of 19.7 kDa, and an optimum pH and temperature for degradation of emulsified PBSA of 7.2 and 45 °C, respectively. Ca²⁺ ion at a concentration of about 1.0 mM markedly enhanced the degradation of emulsified PBSA.     

Transposon-mediated mutation of CYP76AD3 affects betalain synthesis and produces variegated flowers in four o’clock (Mirabilis jalapa)

The variegated flower colors of many plant species have been shown to result from the insertion or excision of transposable elements into genes that encode enzymes involved in anthocyanin synthesis. To date, however, it has not been established whether this phenomenon is responsible for the variegation produced by other pigments such as betalains. During betalain synthesis in red beet, the enzyme CYP76AD1 catalyzes the conversion of l-dihydroxyphenylalanine (DOPA) to cyclo-DOPA. RNA sequencing (RNA-seq) analysis indicated that the homologous gene in four o’clock (Mirabilis jalapa) is CYP76AD3. Here, we show that in four o’clock with red perianths, the CYP76AD3 gene consists of one intron and two exons; however, in a mutant with a perianth showing red variegation on a yellow background, a transposable element, dTmj1, had been excised from the intron. This is the first report that a transposition event affecting a gene encoding an enzyme for betalain synthesis can result in a variegated flower phenotype.     

Role of multidrug resistance-associated protein 1 in the pathogenesis of allergic airway inflammation

Multidrug resistance-associated protein 1 (MRP1) is a cysteinyl leukotriene (CysLT) export pump expressed on mast cells. CysLTs are crucial mediators in allergic airway disease. However, biological significance of MRP1 in allergic airway inflammation has not yet been elucidated. In this study, we sensitized wild-type control mice (mrp1(+/+)) and MRP1-deficient mice (mrp1(-/-)) to ovalbumin (OVA) and challenged them with OVA by aerosol. Airway inflammation and goblet cell hyperplasia after OVA exposure were reduced in mrp1(-/-) mice compared with mrp1(+/+) mice. Furthermore, CysLT levels in bronchoalveolar lavage fluid (BALF) from OVA-exposed mrp1(-/-) mice were significantly lower than those from OVA-exposed mrp1(+/+) mice. Levels of OVA-specific IgE, IL-4, and IL-13 in BALF were also decreased in OVA-exposed mrp1(-/-) mice. IgE-mediated release of CysLTs from murine bone marrow-derived mast cells was markedly impaired by MRP1 deficiency. Our results indicate that MRP1 plays an important role in the development of allergic airway inflammation through regulation of IgE-mediated CysLT export from mast cells.     

Design of stable α‐helices using global sequence optimization

The rational design of peptide and protein helices is not only of practical importance for protein engineering but also is a useful approach in attempts to improve our understanding of protein folding. Recent modifications of theoretical models of helix‐coil transitions allow accurate predictions of the helix stability of monomeric peptides in water and provide new possibilities for protein design. We report here a new method for the design of α‐helices in peptides and proteins using AGADIR, the statistical mechanical theory for helix‐coil transitions in monomeric peptides and the tunneling algorithm of global optimization of multidimensional functions for optimization of amino acid sequences. CD measurements of helical content of peptides with optimized sequences indicate that the helical potential of protein amino acids is high enough to allow formation of stable α‐helices in peptides as short as of 10 residues in length. The results show the maximum achievable helix content (HC) of short peptides with fully optimized sequences at 5 °C is expected to be ～70–75%. Under certain conditions the method can be a powerful practical tool for protein engineering. Unlike traditional approaches that are often used to increase protein stability by adding a few favorable interactions to the protein structure, this method deals with all possible sequences of protein helices and selects the best one from them. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Fatty acids of membrane phospholipids in Drosophila melanogaster lines showing rapid and slow recovery from chill coma

We investigated the fatty acid compositions of phospholipids in Drosophila melanogaster lines showing rapid (CR), intermediate (CTL), or slow (CS) recovery from chill coma, which were established by artificial selection or by free recombination without selection. Compared to CTL, CS showed a low composition of dienoic acids and a small number of double bonds in the fatty acids. The ratio of unsaturated fatty acids and saturated fatty acids (UFAs/SFAs) was significantly lower in CS than in CTL. CR had higher monoenoic acid composition and lower dienoic acid composition than CTL. In addition, the amount of SFAs was lower and therefore the UFAs/SFAs ratio considerably higher in CR than in CTL. These changes in phospholipid fatty acids probably contributed to losing and maintaining the homeoviscosity of the cellular membranes in CS and CR, respectively, at low temperature and therefore produced their distinct phenotypes in recovery from chill coma.