Neurite Outgrowth of PC12 Cells by 4′-O-β-d-Glucopyranosyl-3′,4-Dimethoxychalcone from Brassica rapa L. ‘hidabeni’ was Enhanced by Pretreatment with p38MAPK Inhibitor

The cellular effects of eleven compounds including chalcone glycosides isolated from Brassica rapa L. ‘hidabeni’ and their synthetic derivatives were studied in rat pheochromocytoma PC12 cells. Of the compounds tested, 4′-O-β-d-glucopyranosyl-3′,4-dimethoxychalcone (A2) significantly increased the levels of the phosphorylated forms of extracellular signal-regulated kinases 1/2 (ERK 1/2), p38 mitogen-activated protein kinase (p38MAPK), and stress-activated protein kinases/Jun amino-terminal kinases (JNK/SAPK), but it did not affect Akt. Nerve growth factor (NGF), a well-known neurotrophic factor, increased the levels of phosphorylated ERK1/2, JNK/SAPK, and Akt but not p38MAPK, which may mediate marked neurite outgrowth. Signals evoked by A2 shared common characteristics with those induced by NGF; therefore, we evaluated the neuritogenic activity of A2 and found it induced only weak neurite outgrowth. However, this effect was enhanced by pre-treatment with a p38MAPK inhibitor, suggesting that the phosphorylation of p38MAPK down-regulated neurite outgrowth. From the results of this study, it was found that A2 in combination with a p38MAPK inhibitor can induce NGF-like effects. Hence, a combination of chalcone glycosides containing A2 and a p38MAPK inhibitor increases the likelihood that chalcone glycosides could be put to practical use in the form of drugs or alternative medicines to maintain neural health.     

Rap1 potentiates endothelial cell junctions by spatially controlling myosin II activity and actin organization

Reorganization of the actin cytoskeleton is responsible for dynamic regulation of endothelial cell (EC) barrier function. Circumferential actin bundles (CAB) promote formation of linear adherens junctions (AJs) and tightening of EC junctions, whereas formation of radial stress fibers (RSF) connected to punctate AJs occurs during junction remodeling. The small GTPase Rap1 induces CAB formation to potentiate EC junctions; however, the mechanism underlying Rap1-induced CAB formation remains unknown. Here, we show that myotonic dystrophy kinase–related CDC42-binding kinase (MRCK)-mediated activation of non-muscle myosin II (NM-II) at cell–cell contacts is essential for Rap1-induced CAB formation. Our data suggest that Rap1 induces FGD5-dependent Cdc42 activation at cell–cell junctions to locally activate the NM-II through MRCK, thereby inducing CAB formation. We further reveal that Rap1 suppresses the NM-II activity stimulated by the Rho–ROCK pathway, leading to dissolution of RSF. These findings imply that Rap1 potentiates EC junctions by spatially controlling NM-II activity through activation of the Cdc42–MRCK pathway and suppression of the Rho–ROCK pathway.     

Cryopreservation of rat MSCs by use of a programmed freezer with magnetic field

Mesenchymal stem cells (MSCs) can be used for the regeneration of various tissues and cryopreservation of MSCs is so important for regenerative medicine. The purpose of this study was to evaluate the influences of cryopreservation on MSCs by use of a programmed freezer with a magnetic field (CAS freezer). MSCs were isolated from bone marrow of rat femora. The cells were frozen by a CAS freezer with 10% dimethyl sulfoxide (Me2SO) and cryopreserved for 7 days at a temperature of −150 °C. Immediately after thawing, the number of survived cells was counted. The cell proliferation also examined after 48 h culture. Next, MSCs were frozen by two different freezers; CAS freezer and a conventional programmed freezer without magnetic field. Then, osteogenic and adipogenic differentiations of cryopreserved cells were examined. As a result, survival and proliferation rates of MSCs were significantly higher in CAS freezer than in the non-magnetic freezer. Alizarin positive reaction, large amount of calcium quantification, and greater alkaline phosphatase activity were shown in both the non-cryopreserved and CAS groups after osteogenic differentiation. Moreover, Oil Red O staining positive reaction and high amount of PPARγ and FABP4 mRNAs were shown in both the non-cryopreserved and CAS groups after adipogenic differentiation. From these findings, it is shown that a CAS freezer can maintain high survival and proliferation rates of MSCs and maintain both adipogenic and osteogenic differentiation abilities. It is thus concluded that CAS freezer is available for cryopreservation of MSCs, which can be applied to various tissue regeneration.     

Synthesis and properties of thymidines with six-membered amide bridge

Artificial thymidine monomers possessing amide or N-methylamide bridges were designed, synthesized, and introduced into oligonucleotides. UV-melting experiments showed that these oligonucleotides preferred single-stranded RNA (ssRNA) to single-stranded DNA (ssDNA) in duplex formation. Both amide- and N-methylamide-modified oligonucleotides led to a significant increase in the binding affinity to ssRNA by up to +4.7 and +3.7 °C of the T_m value per modification, respectively, compared with natural oligonucleotide. In addition, their oligonucleotides showed high stability against 3′-exonuclease.     

Glutathione S‐transferase π complexes with and stimulates Na+,K+‐ATPase

Glutathione S‐transferase (GST) was found to complex with the Na⁺,K⁺‐ATPase as shown by binding assay using quartz crystal microbalance. The complexation was obstructed by the addition of antiserum to the α‐subunit of the Na⁺,K⁺‐ATPase, suggesting the specificity of complexation between GST and the Na⁺,K⁺‐ATPase. Co‐immunoprecipitation experiments, using the anti‐α‐subunit antiserum to precipitate the GST‐Na⁺,K⁺‐ATPase complex and then using antibodies specific to an isoform of GST to identify the co‐precipitated proteins, revealed that GSTπ was complexed with the Na⁺,K⁺‐ATPase. GST stimulated the Na⁺,K⁺‐ATPase activity up to 1.4‐fold. The level of stimulation exhibited a saturable dose–response relationship with the amount of GST added, although the level of stimulation varied depending on the content of GSTπ in the lots of GST received from supplier. The stimulation was also obtained when recombinant GSTπ was used, confirming the results. When GST was treated with reduced glutathione, GST activity was greatly stimulated, whereas the level of stimulation of the Na⁺,K⁺‐ATPase activity was similar to that when untreated GST was added. When GST was treated with H₂O₂, GST activity was greatly diminished while the stimulation of the Na⁺,K⁺‐ATPase activity was preserved. The results suggest that GSTπ complexes with the Na⁺,K⁺‐ATPase and stimulates the latter independent of its GST activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Switching of metabolic-rate scaling between allometry and isometry in colonial ascidians

The metabolic rate and its scaling relationship to colony size were studied in the colonial ascidian Botrylloides simodensis. The colonial metabolic rate, measured by the oxygen consumption rate (V(O2) in millilitres of O(2) per hour) and the colony mass (wet weight M(w) in grams) showed the allometric relationship (V(O2) = 0.0412 M(w)(0.799). The power coefficient was statistically not different from 0.75, the value for unitary organisms. The size of the zooids and the tunic volume fraction in a colony were kept constant irrespective of the colonial size. These results, together with the two-dimensional colonial shape, excluded shape factors and colonial composition as possible causes of allometry. Botryllid ascidians show a takeover state in which all the zooids of the parent generation in a colony degenerate and zooids of a new generation develop in unison. The media for connection between zooids such as a common drainage system and connecting vessels to the common vascular system experienced reconstruction. The metabolic rate during the takeover state was halved and was directly proportional to the colonial mass. The scaling thus changed from being allometric to isometric. The alteration in the scaling that was associated with the loss of the connection between the zooids strongly support the hypothesis that the allometry was derived from mutual interaction among the zooids. The applicability of this hypothesis to unitary organisms is discussed.     

Probing the H-protein-induced conformational change and the function of the N-terminal region of Escherichia coli T-protein of the glycine cleavage system by limited proteolysis

T-protein, a component of the glycine cleavage system, catalyzes a tetrahydrofolate-dependent reaction. Previously, we reported a conformational change of Escherichia coli T-protein upon interacting with E. coli H-protein (EH), showing an important role for the N-terminal region of the T-protein in the interaction. To further investigate the T-protein catalysis, the wild type (ET) and mutants were subjected to limited proteolysis. ET was favorably cleaved at Lys(81), Lys(154), Lys(288), and Lys(360) by lysylendopeptidase and the cleavages at Lys(81) and Lys(288) were strongly prevented by EH. Although ET was highly resistant to trypsinolysis, the mutant with an N-terminal 7-residue deletion (ETDelta7) was quite susceptible and instantly cleaved at Arg(16) accompanied by the rapid degradation of the resulting C-terminal fragment, indicating that the cleavage at Arg(16) is the trigger for the C-terminal fragmentation. EH showed no protection from the N-terminal cleavage, although substantial protection from the C-terminal fragmentation was observed. The replacement of Leu(6) of ET with alanine resulted in a similar sensitivity to trypsin as ETDelta7. These results suggest that the N-terminal region of ET functions as a molecular "hasp" to hold ET in the compact form required for the proper association with EH. Leu(6) seems to play a central role in the hasp function. Interestingly, Lys(360) of ET was susceptible to proteolysis even after the stabilization of the entire molecule of ET by EH, indicating its location at the surface of the ET-EH complex. Together with the buried position of Lys(81) in the complex and previous results on folate binding sites, these results suggest the formation of a folate-binding cavity via the interaction of ET with EH. The polyglutamyl tail of the folate substrate may be inserted into the bosom of the cavity leaving the pteridine ring near the entrance of the cavity in the context of the catalytic reaction.     

Imaging of immobilized enzyme spots by scanning chemiluminescence microscopy with electrophoretic injection

Scanning chemiluminescence microscopy (SCLM) with electrophoretic injection was developed and applied to visualize enzyme reactions localized in an enzyme microspot. The SCLM uses a tapered glass capillary as a probe for injecting a small amount of luminol onto the substrate to generate localized chemiluminescence. The electrophoretic injection by application of a constant current between the inside and outside of the capillary enabled the continuous and controllable injection of a minute quantity of luminol in the range of 0.1 pmol/s. The image of enzyme activity in a monolayer spot of horseradish peroxidase was obtained by using the electrophoretic injection-based SCLM system.     

Cirri of the stalked crinoid Metacrinus rotundus: neural elements and the effect of cholinergic agonists on mechanical properties

Sea lilies are enigmatic animals due to their scarcity and their biology is comparatively neglected. Cirri, arranged in whorls of five along the sea lily stalk, anchor and support the animal. They consist of ossicles interconnected by collagenous ligaments and by a central canal. Cirri have a well-developed nervous system but lack muscular cells. A light and electron microscopic study was performed to clarify the morphology of the nervous system of the cirri. Two cellular networks were found, one of neuron-like cells and one of cells filled with bullet-shaped organelles. Both networks ramify throughout the cirral ossicles up to the interossicle ligaments. Mechanical tests were performed to analyse the influence of cholinergic agonists on the mechanical properties of these ligaments. In the tests, the cirral ligaments softened after the application of acetylcholine, muscarinic agonists and nicotinic agonists. The reaction time to muscarinic agonists was much slower than to acetylcholine and nicotinic agonists. At low concentrations, muscarinic agonists caused active development of force. No reaction to stimuli was observed in anaesthetized cirri. The data clearly establish the existence of catch connective tissue which can change its mechanical properties under nervous control mediated via nerves with cholinergic receptors. The possible sources of the observed force production are discussed and it is concluded that active contraction of collagenous ligaments causes movement of cirri.