Surface modification of bacterial cellulose nanofibers for property enhancement of optically transparent composites: dependence on acetyl-group DS

Bacterial cellulose (BC) nanofibers were acetylated to enhance the properties of optically transparent composites of acrylic resin reinforced with the nanofibers. A series of BC nanofibers acetylated from degree-of-substitution (DS) 0 to 1.76 were obtained. X-ray diffraction profiles indicated that acetylation proceeded from the surface to the core of BC nanofibers, and scanning electron microscopy images showed that the volume of nanofibers increases by the bulky acetyl group. Since acetylation decreased the refractive index of cellulose, regular transmittance of composites comprised of 63% BC nanofiber was improved, and deterioration at 580 nm because of fiber reinforcement was suppressed to only 3.4%. Acetylation of nanofibers changed their surface properties and reduced the moisture content of the composite to about one-third that of untreated composite, although excessive acetylation increased hygroscopicity. Furthermore, acetylation was found to reduce the coefficient of thermal expansion of a BC sheet from 3 x 10(-6) to below 1 x 10(-6) 1/K.     

Exploration of Pericyte-Derived Factors Implicated in Lung Cancer Brain Metastasis Protection: A Pilot Messenger RNA Sequencing Using the Blood–Brain Barrier In Vitro Model

Cellular and Molecular Neurobiology - Metastatic brain tumors have poor prognoses and pose unmet clinical problems for the patients. The blood–brain barrier (BBB) implication is supposed to...     

Reexamination of Chlorophyllase Function Implies Its Involvement in Defense against Chewing Herbivores

Chlorophyllase (CLH) is a common plant enzyme that catalyzes the hydrolysis of chlorophyll to form chlorophyllide, a more hydrophilic derivative. For more than a century, the biological role of CLH has been controversial, although this enzyme has been often considered to catalyze chlorophyll catabolism during stress-induced chlorophyll breakdown. In this study, we found that the absence of CLH does not affect chlorophyll breakdown in intact leaf tissue in the absence or the presence of methyl-jasmonate, which is known to enhance stress-induced chlorophyll breakdown. Fractionation of cellular membranes shows that Arabidopsis (Arabidopsis thaliana) CLH is located in the endoplasmic reticulum and the tonoplast of intact plant cells. These results indicate that CLH is not involved in endogenous chlorophyll catabolism. Instead, we found that CLH promotes chlorophyllide formation upon disruption of leaf cells, or when it is artificially mistargeted to the chloroplast. These results indicate that CLH is responsible for chlorophyllide formation after the collapse of cells, which led us to hypothesize that chlorophyllide formation might be a process of defense against chewing herbivores. We found that Arabidopsis leaves with genetically enhanced CLH activity exhibit toxicity when fed to Spodoptera litura larvae, an insect herbivore. In addition, purified chlorophyllide partially suppresses the growth of the larvae. Taken together, these results support the presence of a unique binary defense system against insect herbivores involving chlorophyll and CLH. Potential mechanisms of chlorophyllide action for defense are discussed.Plants have evolved both constitutive and inducible defense mechanisms against herbivores. Constitutive mechanisms include structural defenses (e.g. spines and trichomes) and specific chemical compounds. Constitutive defense mechanisms provide immediate protection against herbivore attacks, although they represent an energy investment by the plant regardless of whether herbivory occurs or not (Mauricio, 1998; Bekaert et al., 2012). By contrast, inducible defense mechanisms do not require an up-front energy cost, although such mechanisms may not be as immediate as constitutive ones when herbivore feeding occurs (Windram et al., 2012). Accordingly, plants exhibit both constitutive and inducible defense mechanisms against herbivory to balance the speed and cost of response. In this regard, it is plausible that the recruitment of abundant primary metabolites for defensive purposes might represent a substantial benefit to plants, providing both a swift and economical defense function.Toxic chemical compounds form an essential part in both constitutive and inducible defense mechanisms. However, these compounds are potentially a double-edged sword for plants, in a sense that they might pose toxic effects for both plants and herbivores. Plants have evolved an intricate binary system that prevents autointoxication by their own chemical compounds. Specifically, a toxic substance is stored in its inactive form and is spatially isolated from specific activating enzymes. These enzymes activate the substance when cells are disrupted by chewing herbivores (Saunders and Conn, 1978; Thayer and Conn, 1981; Morant et al., 2008). One of the most extensively studied binary defense systems is the glucosinolate/myrosinase system, in which the glucosinolate substrate and their hydrolyzing enzyme, a thioglucosidase myrosinase, are compartmentalized. Upon tissue damage, both the substrate and the enzyme come into contact to produce unstable aglycones, and various toxic compounds are then spontaneously produced (Bones and Rossiter, 1996). Another well-known example of the binary system is comprised of cyanogenic glucosides and β-glucosidase (Vetter, 2000; Mithöfer and Boland, 2012). In this system, nontoxic cyanogenic glycoside compounds are stored in the vacuole, whereas, the related glycosidase is localized in the cytoplasm. Upon cell destruction by chewing herbivores, the cyanogenic glycosides are hydrolyzed by glycosidase to yield unstable cyanohydrin that is either spontaneously or enzymatically converted into toxic hydrogen cyanide and a ketone or an aldehyde. Because the binary defense system is efficient and effective, a use of ubiquitous compounds for such systems would provide further benefits for plants.Tetrapyrrole compounds, in particular heme and chlorophyll, are abundant in plant cells. Despite their significant roles in various biological processes including photosynthesis and respiration, many tetrapyrroles are highly toxic to plant and animal cells, if present in excess amounts (Kruse et al., 1995; Meskauskiene et al., 2001). Their photodynamic properties can cause the generation of reactive oxygen species upon illumination, resulting in cell injury or direct cell death. For example, Tapper et al. (1975) showed that a tetrapyrrole compound (pheophorbide a), which is readily converted from dietary chlorophyll through the loss of magnesium and phytol, reduces the growth and survival rates of young albino rats through its photodynamic property. More recently, Jonker et al. (2002) demonstrated that dietary-derived pheophorbide a causes severe damages on the skin of mutant mice that lack a transporter to excrete pheophorbide a from cells. These studies indicate that incorporation of an excessive amount of tetrapyrrole compounds can induce photosensitization in animals. Previous studies also showed that tetrapyrroles have illumination-independent deleterious effects on insects. For example, pheophorbide a affected the assimilation of the plant sterols to synthesize developmental hormones of insects by inhibiting the activity of a key enzyme, cholesterol acyltransferase (Song et al., 2002). Moreover, some tetrapyrroles, including pheophorbide a, have been suggested to induce illumination-independent cell death in plants as well by an unknown mechanism (Hirashima et al., 2009). It is proposed that organisms use the toxicity of tetrapyrroles for their defense systems. The larvae of tortoise beetle (Chelymorpha alternans) even utilize pheophorbide a as a powerful deterrent in the fecal shield to protect themselves from their predators (Vencl et al., 2009). Kariola et al. (2005) suggested that a chlorophyll derivative, chlorophyllide, is involved in the defense against fungi, based on their observations that down-regulation of a chlorophyll-hydrolyzing enzyme, chlorophyllase (CLH), results in increased susceptibility of Arabidopsis (Arabidopsis thaliana) plants to the necrotrophic fungus Alternaria brassicicola.In this study, we examined the possibility that plants use tetrapyrroles for defense against herbivores by analyzing CLH, a well-known hydrolase common in plants. Chlorophyll consists of a tetrapyrrolic macrocycle and a hydrophobic phytol side chain (Fig. 1). Phytol hydrolysis results in the formation of chlorophyllide (Fig. 1), a less hydrophobic chlorophyll derivative, which has photochemical properties similar to chlorophyll. Two different plant enzymes are known to catalyze the cleavage of phytol, pheophytinase (PPH) and CLH. PPH is a chloroplast-located enzyme that specifically catalyzes the removal of phytol from Mg-free chlorophyll catabolites (Schelbert et al., 2009). This enzyme was only recently discovered and has been shown to be responsible for chlorophyll degradation during leaf senescence. By contrast, CLH has a broader substrate specificity and removes the side chain from chlorophyll or other chlorophyll derivatives (McFeeters et al., 1971). CLH activity was first reported in leaf extracts in 1913 (Willstätter and Stoll, 1913), but despite a century of research, in vivo function and intracellular localization of this enzyme remained controversial. Some reports have indicated CLH to localize to chloroplasts (Azoulay Shemer et al., 2008; Azoulay-Shemer et al., 2011), while Schenk et al. (2007), by examining the intracellular localization of transiently expressed CLH-GFP fusions, proposed Arabidopsis CLH to localize outside the chloroplast. Schenk et al. (2007) also reported that the lack of CLH does not affect chlorophyll degradation during leaf senescence. However, it remains possible that CLH is specifically involved in chlorophyll degradation in response to stresses that activate jasmonate signaling, such as wounding or pathogen attack. This hypothesis is based on the observation that the expression of a CLH gene was highest when methyl-jasmonate (MeJA; a derivative of jasmonic acid) was applied to Arabidopsis plants (Tsuchiya et al., 1999).Open in a separate windowFigure 1.Early steps of proposed chlorophyll breakdown pathways. MCS, Magnesium-dechelating substance.Here, we report that CLH is not involved in endogenous chlorophyll breakdown even when leaf senescence was promoted by jasmonate signaling. CLH is shown to localize to the chlorophyll-free endoplasmic reticulum (ER) and the tonoplast of intact plant cells. We found that CLH promotes the conversion of chlorophyll into chlorophyllide when leaf cells are disrupted or when CLH is genetically mislocalized to chloroplasts. To examine the possibility that plants use chlorophyll and CLH to form a binary defense system against herbivores, a generalist herbivore, Spodoptera litura larvae, was employed to investigate the toxicity of Arabidopsis leaves with genetically enhanced CLH activity and purified chlorophyllide. The results support our hypothesis, indicating plants to deploy an abundant photosynthetic pigment for defense against herbivores, which would be economic and provide adaptation benefits to plants. A potential mechanism of chlorophyllide action as part of the plant defense system is discussed based on the examination of chlorophyllide binding to the insect gut.     

Effective Trapping of Fruit Flies with Cultures of Metabolically Modified Acetic Acid Bacteria

Acetoin in vinegar is an attractant to fruit flies when combined with acetic acid. To make vinegar more effective in attracting fruit flies with increased acetoin production, Komagataeibacter europaeus KGMA0119 was modified by specific gene disruption of the acetohydroxyacid isomeroreductase gene (ilvC). A previously constructed mutant lacking the putative ligand-sensing region in the leucine-responsive regulatory protein (KeLrp, encoded by Kelrp) was also used. The ilvC and Kelrp disruptants (KGMA5511 and KGMA7203, respectively) produced greater amounts of acetoin (KGMA5511, 0.11%; KGMA7203, 0.13%) than the wild-type strain KGMA0119 (0.069%). KGMA7203 produced a trace amount of isobutyric acid (0.007%), but the other strains did not. These strains produced approximately equal amounts of acetic acid (0.7%). The efficiency of fruit fly attraction was investigated with cultured Drosophila melanogaster. D. melanogaster flies (approximately 1,500) were released inside a cage (2.5 m by 2.5 m by 1.5 m) and were trapped with a device containing vinegar and a sticky sheet. The flies trapped on the sticky sheet were counted. The cell-free supernatant from KGMA7203 culture captured significantly more flies (19.36 to 36.96% of released flies) than did KGMA0119 (3.25 to 11.40%) and KGMA5511 (6.87 to 21.50%) cultures. Contrastingly, a 0.7% acetic acid solution containing acetoin (0.13%) and isobutyric acid (0.007%), which mimicked the KGMA7203 supernatant, captured significantly fewer flies (0.88 to 4.57%). Furthermore, the KGMA0119 supernatant with additional acetoin (0.13%) and isobutyric acid (0.007%) captured slightly more flies than the original KGMA0119 supernatant but fewer than the KGMA7203 supernatant, suggesting that the synergistic effects of acetic acid, acetoin, isobutyric acid, and unidentified metabolites achieved the efficient fly trapping of the KGMA7203 supernatant.     

Plasma gelsolin facilitates interaction between β2 glycoprotein I and α5β1 integrin

Antiphospholipid syndrome (APS) is characterized by thrombosis and the presence of antiphospholipid antibodies (aPL) that directly recognizes plasma β₂‐glycoprotein I (β₂GPI). Tissue factor (TF), the major initiator of the extrinsic coagulation system, is induced on monocytes by aPL in vitro, explaining in part the pathophysiology in APS. We previously reported that the mitogen‐activated protein kinase (MAPK) pathway plays an important role in aPL‐induced TF expression on monocytes. In this study, we identified plasma gelsolin as a protein associated with β₂GPI by using immunoaffinity chromatography and mass spectrometric analysis. An in vivo binding assay showed that endogenous β₂GPI interacts with plasma gelsolin, which binds to integrin a₅β₁ through fibronectin. The tethering of β₂GPI to monoclonal anti‐β₂GPI autoantibody on the cell surface was enhanced in the presence of plasma gelsolin. Immunoblot analysis demonstrated that p38 MAPK protein was phosphorylated by monoclonal anti‐β₂GPI antibody treatment, and its phosphorylation was attenuated in the presence of anti‐integrin a₅β₁ antibody. Furthermore, focal adhesion kinase, a downstream molecule of the fibronectin‐integrin signalling pathway, was phosphorylated by anti‐β₂GPI antibody treatment. These results indicate that molecules including gelsolin and integrin are involved in the anti‐β₂GPI antibody‐induced MAPK pathway on monocytes and that integrin is a possible therapeutic target to modify a prothrombotic state in patients with APS.     

Correlations among brain gray matter volumes, age, gender, and hemisphere in healthy individuals

To determine the relationship between age and gray matter structure and how interactions between gender and hemisphere impact this relationship, we examined correlations between global or regional gray matter volume and age, including interactions of gender and hemisphere, using a general linear model with voxel-based and region-of-interest analyses. Brain magnetic resonance images were collected from 1460 healthy individuals aged 20-69 years; the images were linearly normalized and segmented and restored to native space for analysis of global gray matter volume. Linearly normalized images were then non-linearly normalized and smoothed for analysis of regional gray matter volume. Analysis of global gray matter volume revealed a significant negative correlation between gray matter ratio (gray matter volume divided by intracranial volume) and age in both genders, and a significant interaction effect of age × gender on the gray matter ratio. In analyzing regional gray matter volume, the gray matter volume of all regions showed significant main effects of age, and most regions, with the exception of several including the inferior parietal lobule, showed a significant age × gender interaction. Additionally, the inferior temporal gyrus showed a significant age × gender × hemisphere interaction. No regional volumes showed significant age × hemisphere interactions. Our study may contribute to clarifying the mechanism(s) of normal brain aging in each brain region.     

Disruption of Sept6, a fusion partner gene of MLL, does not affect ontogeny, leukemogenesis induced by MLL-SEPT6, or phenotype induced by the loss of Sept4

Septins are evolutionarily conserved GTP-binding proteins that can heteropolymerize into filaments. Recent studies have revealed that septins are involved in not only diverse normal cellular processes but also the pathogenesis of various diseases, including cancer. SEPT6 is ubiquitously expressed in tissues and one of the fusion partner genes of MLL in the 11q23 translocations implicated in acute leukemia. However, the roles of this septin in vivo remain elusive. We have developed Sept6-deficient mice that exhibited neither gross abnormalities, changes in cytokinesis, nor spontaneous malignancy. Sept6 deficiency did not cause any quantitative changes in any of the septins evaluated in this study, nor did it cause any additional changes in the Sept4-deficient mice. Even the depletion of Sept11, a close homolog of Sept6, did not affect the Sept6-null cells in vitro, thus implying a high degree of redundancy in the septin system. Furthermore, a loss of Sept6 did not alter the phenotype of myeloproliferative disease induced by MLL-SEPT6, thus suggesting that Sept6 does not function as a tumor suppressor. To our knowledge, this is the first report demonstrating that a disruption of the translocation partner gene of MLL in 11q23 translocation does not contribute to leukemogenesis by the MLL fusion gene.