Interaction between avoidance of photon absorption,excess energy dissipation and zeaxanthin synthesis against photooxidative stress in Arabidopsis

Plants evolved photoprotective mechanisms in order to counteract the damaging effects of excess light in oxygenic environments. Among them, chloroplast avoidance and non‐photochemical quenching concur in reducing the concentration of chlorophyll excited states in the photosynthetic apparatus to avoid photooxidation. We evaluated their relative importance in regulating excitation pressure on photosystem II. To this aim, genotypes were constructed carrying mutations impairing the chloroplast avoidance response (phot2) as well as mutations affecting the biosynthesis of the photoprotective xanthophyll zeaxanthin (npq1) or the activation of non‐photochemical quenching (npq4), followed by evaluation of their photosensitivity in vivo. Suppression of avoidance response resulted in oxidative stress under excess light at low temperature, while removing either zeaxanthin or PsbS had a milder effect. The double mutants phot2 npq1 and phot2 npq4 showed the highest sensitivity to photooxidative stress, indicating that xanthophyll cycle and qE have additive effects over the avoidance response. The interactions between non‐photochemical quenching and avoidance responses were studied by analyzing the kinetics of fluorescence decay and recovery at different light intensities. phot2 fluorescence decay lacked a component, here named as qM. This kinetic component linearly correlated with the leaf transmittance changes due to chloroplast relocation induced by white light and was absent when red light was used as actinic source. On these basis we conclude that a decrease in leaf optical density affects the apparent non‐photochemical quenching (NPQ) rise kinetic. Thus, excess light‐induced fluorescence decrease is in part due to avoidance of photon absorption rather than to a genuine quenching process.     

The effect of 17-N substituents on the activity of the opioid κ receptor in nalfurafine derivatives

We have previously reported the essential structure of the opioid κ receptor agonist nalfurafine hydrochloride (TRK-820) for binding to the κ receptor. In the course of this study, we focused on the effect of the substituent at 17-N in nalfurafine on the binding affinity for the κ receptor. The exchange of the 17-N substituent in nalfurafine from cyclopropylmethyl to fluoro-substituted alkyl groups, which are strong electron withdrawing substituents, almost completely diminished the binding affinities for the μ and δ opioid receptors, but the binding affinity for the κ receptor was still maintained. As a result, nalfurafine derivatives with 17-fluoro-substituted alkyl groups showed higher selectivities for the κ receptor than did nalfurafine itself. With regard to the κ agonistic activities, the conversion of the 17-N substituent in nalfurafine from cyclopropylmethyl to fluoro-substituted alkyl groups led to the gradual decrease of the agonistic activities in the order corresponding to their binding affinities for the κ receptor. In contrast, the derivative with the bulky 17-isobutyl group showed lower affinity and agonistic activity for the κ receptor than the derivatives with the smaller functional groups. This research suggested that both the electronic property and the steric characteristics of the 17-N substituent would have a great influence on the binding property for the κ receptor.     

Effect of Ala replacement with Aib in amphipathic cell-penetrating peptide on oligonucleotide delivery into cells

A number of cell-penetrating peptides (CPPs) have been characterized and their usefulness as delivery tools has been clarified. As one of the CPPs, model amphipathic peptide (MAP) was developed by integrating both hydrophobic and hydrophilic amino acids in its sequence. In our previous work, we designed MAP(Aib) by replacing five alanine (Ala) residues on the hydrophobic face of the helix in the MAP sequence with α-aminoisobutyric acid (Aib) residues, and the replacement resulted in higher helix propensity, stronger resistance to protease, and higher cell membrane permeability than MAP. As a next step, we examined the efficiency of oligonucleotide (ODN) delivery into cells by MAP(Aib) in comparison with that by MAP. The electrostatically formed MAP(Aib)/ODN complex was more easily taken up by cells than the MAP/ODN complex, and the ODN delivery by MAP(Aib) was via an endocytic pathway. We demonstrated that the incorporation of Aib residues into CPPs enhances the delivery of hydrophilic molecules, such as ODN, into cells.     

Parkinson's disease-associated mutations in α-synuclein and UCH-L1 inhibit the unconventional secretion of UCH-L1

Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) is an intracellular protein abundantly expressed in neurons, and a mutation in UCH-L1 has been identified in familial Parkinson’s disease. UCH-L1 has been detected in human cerebrospinal fluid, raising the possibility that UCH-L1 is secreted from neurons. In the present study, we showed that a portion of UCH-L1 is secreted from cultured cells. The secretion of D30K UCH-L1, which lacks ubiquitin binding activity, was decreased compared with that of wild-type UCH-L1, while the secretion of C90S UCH-L1, which lacks hydrolase activity, was not. Treatment with Brefeldin A, an inhibitor of vesicle transport from the endoplasmic reticulum to the Golgi, did not block the secretion of UCH-L1, indicating that UCH-L1 is secreted by an unconventional pathway. The UCH-L1 sequence from Leu-32 to Leu-39 is similar to the unconventional secretory signal sequence of engrailed 2, and substitution of the leucines within this region (L32S/L32A/L34S/L34A/L39S/L39A) reduced the secretion of UCH-L1. We found that the Parkinson’s disease-associated mutation I93M in UCH-L1 decreased the secretion of I93M UCH-L1. In addition, Parkinson’s disease-linked α-synuclein mutants reduced the secretion of endogenous UCH-L1. Our results indicate that the hydrolase activity is not necessary for the unconventional secretion of UCH-L1, and suggest that the ubiquitin binding activity and the sequence between Leu-32 and Leu-39 are involved in the secretion. Moreover, the secretion of UCH-L1 could be involved in the pathology of Parkinson’s disease.     

Analysis of IIId, IIIe and IVa group basic-helix-loop-helix proteins expressed in Arabidopsis root epidermis

Differentiation of Arabidopsis epidermal cells into root hairs and trichomes is a functional model system for understanding plant cell development. Previous studies showed that one of the Arabidopsis basic-helix-loop-helix (AtbHLH) proteins, GLABRA3 (GL3), is involved in root-hair and trichome differentiation. We analyzed 11 additional AtbHLH genes with homology to GL3. Estimation of the phylogeny based on amino acid sequences of the bHLH region suggests that 11 AtbHLH genes used in this study evolved by duplications of a single common GL3 ancestor. Promoter-GUS analysis showed that AtbHLH006, AtbHLH013, AtbHLH017 and AtbHLH020 were expressed in roots. Among them, AtbHLH006 and AtbHLH020 were preferentially expressed in root epidermal non-hair cells. Consistent with the expression patterns from promoter-GUS analysis, GFP fluorescence was observed in the nuclei of root epidermal non-hair cells of AtbHLH006p::AtbHLH006:GFP and AtbHLH020p::AtbHLH020:GFP transgenic plants. However, AtbHLH006 and AtbHLH0020 proteins did not interact with epidermis-specific MYB proteins and TTG1. Taken together, AtbHLH006 and AtbHLH020 may function in root epidermal cells, but other GL3-like bHLH proteins may have evolved to regulate different processes.     

Gravity-dependent changes in bioconvection of Tetrahymena and Chlamydomonas during parabolic flight: increases in wave number induced by pre- and post-parabola hypergravity

Bioconvection emerges in a dense suspension of swimming protists as a consequence of their negative-gravitactic upward migration and later settling as a blob of density greater than that of water. Thus, gravity is an important parameter governing bioconvective pattern formation. However, inconsistencies are found in previous studies dealing with the response of bioconvection patterns to increased gravity acceleration (hypergravity); the wave number of the patterns has been reported to decrease during the hypergravity phases of parabolic aircraft flight, while it increases in centrifugal hypergravity. In this paper, we reassess the responses of bioconvection to altered gravity during parabolic flight on the basis of vertical and horizontal observations of the patterns formed by Tetrahymena thermophila and Chlamydomonas reinhardtii. Spatiotemporal analyses of the horizontal patterns revealed an increase in the pattern wave number in both pre- and post-parabola hypergravity. Vertical pattern analysis was generally in line with the horizontal pattern analysis, and further revealed that hypergravity-induced changes preceded at the top layer of the suspensions while microgravity-induced changes appeared to occur from the bottom part of the settling blobs. The responses to altered gravity were rather different between the two sample species: T. thermophila tended to drastically modify its bioconvection patterns in response to changes in gravity level, while the patterns of C. reinhardtii responded to a much lesser extent. This difference can be attributed to the distinct physical and physiological properties of the individual organisms, suggesting a significant contribution of the gyrotactic property to the swimming behavior of some protists.     

Ceruloplasmin Protects Against Rotenone-Induced Oxidative Stress and Neurotoxicity

To clarify the neuroprotective property of ceruloplasmin and the pathogenesis of aceruloplasminemia, we generated ceruloplasmin-deficient (CP ^−/−) mice on the C57BL/10 genetic background and further treated them with a mitochondrial complex I inhibitor, rotenone. There was no iron accumulation in the brains of CP ^−/− mice at least up to 60 weeks of age. Without rotenone treatment, CP ^−/− mice showed slight motor dysfunction compared with CP ^+/+ mice, but there were no detectable differences in the levels of oxidative stress markers between these two groups. A low dose of rotenone did not affect the mitochondrial complex I activity in our mice, however, it caused a significant change in motor behavior, neuropathology, or the levels of oxidative stress markers in CP ^−/− mice, but not in CP ^+/+ mice. Our data support that ceruloplasmin protects against rotenone-induced oxidative stress and neurotoxicity, probably through its antioxidant properties independently of its function of iron metabolism.     

Hippo pathway regulation by cell morphology and stress fibers

The Hippo signaling pathway plays an important role in regulation of cell proliferation. Cell density regulates the Hippo pathway in cultured cells; however, the mechanism by which cells detect density remains unclear. In this study, we demonstrated that changes in cell morphology are a key factor. Morphological manipulation of single cells without cell-cell contact resulted in flat spread or round compact cells with nuclear or cytoplasmic Yap, respectively. Stress fibers increased in response to expanded cell areas, and F-actin regulated Yap downstream of cell morphology. Cell morphology- and F-actin-regulated phosphorylation of Yap, and the effects of F-actin were suppressed by modulation of Lats. Our results suggest that cell morphology is an important factor in the regulation of the Hippo pathway, which is mediated by stress fibers consisting of F-actin acting upstream of, or on Lats, and that cells can detect density through their resulting morphology. This cell morphology (stress-fiber)-mediated mechanism probably cooperates with a cell-cell contact (adhesion)-mediated mechanism involving the Hippo pathway to achieve density-dependent control of cell proliferation.