Direct control of exocytosis by receptor-mediated activation of the heterotrimeric GTPases Gi and G(o) or by the expression of their active G alpha subunits.

The exocytotic release of potent hormones is a tightly controlled process. Its direct regulation without the involvement of second messengers would ensure rapid signal processing. In streptolysin O-permeabilized insulin-secreting cells, a preparation allowing dialysis of cytosolic macromolecules, activation of alpha 2-adrenergic receptors caused pertussis toxin-sensitive inhibition of calcium-induced exocytosis. This inhibition was mimicked very efficiently by the use of specific receptor-mimetic peptides, indicating the involvement of Gi and, to a lesser extent, of G(o). The regulation was exerted beyond the ATP-dependent step of exocytosis. In addition, low nanomolar amounts of pre-activated Gi/G(o) directly inhibited exocytosis. As transient overexpression of constitutively active mutants of G alpha i1, G alpha i2, G alpha i3 and G alpha o2 but not of G alpha o1 reproduced this regulation, the G alpha subunit alone is sufficient to induce inhibition. These results define exocytosis as an effector for heterotrimeric G-proteins and delineate the properties of the transduction pathway.     

Downregulation of p‐COUMAROYL ESTER 3‐HYDROXYLASE in rice leads to altered cell wall structures and improves biomass saccharification

p‐Coumaroyl ester 3‐hydroxylase (C3′H) is a key enzyme involved in the biosynthesis of lignin, a phenylpropanoid polymer that is the major constituent of secondary cell walls in vascular plants. Although the crucial role of C3′H in lignification and its manipulation to upgrade lignocellulose have been investigated in eudicots, limited information is available in monocotyledonous grass species, despite their potential as biomass feedstocks. Here we address the pronounced impacts of C3′H deficiency on the structure and properties of grass cell walls. C3′H‐knockdown lines generated via RNA interference (RNAi)‐mediated gene silencing, with about 0.5% of the residual expression levels, reached maturity and set seeds. In contrast, C3′H‐knockout rice mutants generated via CRISPR/Cas9‐mediated mutagenesis were severely dwarfed and sterile. Cell wall analysis of the mature C3′H‐knockdown RNAi lines revealed that their lignins were largely enriched in p‐hydroxyphenyl (H) units while being substantially reduced in the normally dominant guaiacyl (G) and syringyl (S) units. Interestingly, however, the enrichment of H units was limited to within the non‐acylated lignin units, with grass‐specific γ‐p‐coumaroylated lignin units remaining apparently unchanged. Suppression of C3′H also resulted in relative augmentation in tricin residues in lignin as well as a substantial reduction in wall cross‐linking ferulates. Collectively, our data demonstrate that C3′H expression is an important determinant not only of lignin content and composition but also of the degree of cell wall cross‐linking. We also demonstrated that C3′H‐suppressed rice displays enhanced biomass saccharification.     

Detection of G protein-activator regions in M4 subtype muscarinic, cholinergic, and alpha 2-adrenergic receptors based upon characteristics in primary structure.

The 14-residue region Arg2410-Lys2423 of the human insulin-like growth factor II receptor possesses the ability to stimulate Gi, the activity being dependent on two structural characteristics: (i) at least two basic residues at the N-terminal side and (ii) the C-terminal motif, B-B-X-B or B-B-X-X-B (where B is a basic residue and X is a non-basic residue). The regions satisfying (i) and (ii) with 10 less than or equal to residue length less than or equal to 26 were located in all of the third inner loops and some of the other intracellular domains of the Gi-coupled M4 sub-type muscarinic cholinergic receptor (M4AChR) and the alpha 2-adrenergic receptor (alpha 2AR). Both the second inner loop 130-147 and the C-terminal portion of the third inner loop 382-400 (MIII) of human M4AChR had the ability to stimulate G proteins with the order Gi approximately Go greater than Gs, but only MIII could activate Gi/Go at nanomolar concentrations. In contrast, the N-terminal portion of the third inner loop 218-228 of human alpha 2AR-C10 activated Gi, Go, and Gs at micromolar concentrations with equal potency, whereas the further C-terminal portion of the third inner loop 301-313 of this receptor lacked the ability to activate any G protein. Among these active regions, only MIII indicated Mg(2+)-dependent Gi-stimulating function. Therefore, the search for the regions satisfying (i) and (ii) was useful to localize the G protein-activating activity of Gi-coupled receptors in limited regions, which were not always in the C-terminal portions of the third intracellular loops and activated G proteins in various modes of actions.     

A Dictyostelium cellobiohydrolase orthologue that affects developmental timing

Dictyostelium discoideum is a facultative multicellular amoebozoan with cellulose in the stalk and spore coat of its fruiting body as well as in the extracellular matrix of the migrating slug. The organism also harbors a number of cellulase genes. One of them, cbhA, was identified as a candidate cellobiohydrolase gene based on the strong homology of its predicted protein product to fungal cellobiohydrolase I (CBHI). Expression of the cbhA was developmentally regulated, with strong expression in the spores of the mature fruiting body. However, a weak but detectable level of expression was observed in the extracellular matrix at the mound — tipped finger stages, in prestalk O cells, and in the slime sheath of the migrating slug — late culminant stages. A null mutant of the cbhA showed almost normal morphology. However, the developmental timing of the mutant was delayed by 2–4 h. When a c-Myc epitope-tagged CbhA was expressed, it was secreted into the culture medium and was able to bind crystalline cellulose. The CbhA-myc protein was glycosylated, as demonstrated by its ability to bind succinyl concanavalin A-agarose. Moreover, conditioned medium from the cbhA-myc ^oe strain displayed 4-methylumbelliferyl β-d-cellobioside (4-MUC) digesting activity in Zymograms in which conditioned medium was examined via native-polyacrylamide gel electrophoresis or spotted on an agar plate containing 4-MUC, one of the substrates of cellobiohydrolase. Taken together, these findings indicate that Dictyostelium CbhA is an orthologue of CBH I that is required for a normal rate of development.     

Autophagy-related protein 32 acts as autophagic degron and directly initiates mitophagy

Autophagy-related degradation selective for mitochondria (mitophagy) is an evolutionarily conserved process that is thought to be critical for mitochondrial quality and quantity control. In budding yeast, autophagy-related protein 32 (Atg32) is inserted into the outer membrane of mitochondria with its N- and C-terminal domains exposed to the cytosol and mitochondrial intermembrane space, respectively, and plays an essential role in mitophagy. Atg32 interacts with Atg8, a ubiquitin-like protein localized to the autophagosome, and Atg11, a scaffold protein required for selective autophagy-related pathways, although the significance of these interactions remains elusive. In addition, whether Atg32 is the sole protein necessary and sufficient for initiation of autophagosome formation has not been addressed. Here we show that the Atg32 IMS domain is dispensable for mitophagy. Notably, when anchored to peroxisomes, the Atg32 cytosol domain promoted autophagy-dependent peroxisome degradation, suggesting that Atg32 contains a module compatible for other organelle autophagy. X-ray crystallography reveals that the Atg32 Atg8 family-interacting motif peptide binds Atg8 in a conserved manner. Mutations in this binding interface impair association of Atg32 with the free form of Atg8 and mitophagy. Moreover, Atg32 variants, which do not stably interact with Atg11, are strongly defective in mitochondrial degradation. Finally, we demonstrate that Atg32 forms a complex with Atg8 and Atg11 prior to and independent of isolation membrane generation and subsequent autophagosome formation. Taken together, our data implicate Atg32 as a bipartite platform recruiting Atg8 and Atg11 to the mitochondrial surface and forming an initiator complex crucial for mitophagy.     

ALDH2 polymorphism for the risk of cervical carcinogenesis

To investigate the clinical significance of ALDH2 genetic polymorphisms in cervical carcinogenesis. ALDH2 polymorphisms together with human papillomavirus (HPV) types were examined in a total of 195 cervical smear in exfoliated cervical cell samples using Real-Time polymerase chain reaction (PCR) System. The frequency for the AG+AA genotype was seven in the normal group (70.0 %), 16 in the LSIL group (57.1 %), and 27 in the HSIL group (90.0 %). A significant difference was found between the LSIL and HSIL groups (P = 0.0064). Patients with HSIL lesions frequently had high-risk HPV infections and concurrently belonged to the AG+AA group. ALDH2 genotype in cervical cell samples may be associated with more severe precancerous lesions of the cervix in a Japanese population.     

Wortmannin, a PI3-kinase inhibitor: promoting effect on insulin secretion from pancreatic beta cells through a cAMP-dependent pathway

To determine the role of phosphatidylinositol 3-kinase (PI3-kinase) in the regulation of insulin secretion, we examined the effect of wortmannin, a PI3-kinase inhibitor, on insulin secretion using the isolated perfused rat pancreas and freshly isolated islets. In the perfused pancreas, 10(-8) M wortmannin significantly enhanced the insulin secretion induced by the combination of 8.3 mM glucose and 10(-5) M forskolin. In isolated islets, cyclic AMP (cAMP) content was significantly increased by wortmannin in the presence of 3.3 mM, 8.3 mM, and 16.7 mM glucose with or without forskolin. In the presence of 16.7 mM glucose with or without forskolin, wortmannin promoted insulin secretion significantly. On the other hand, in the presence of 8.3 mM glucose with forskolin, wortmannin augmented insulin secretion significantly; although wortmannin tended to promote insulin secretion in the presence of glucose alone, it was not significant. To determine if wortmannin increases cAMP content by promoting cAMP production or by inhibiting cAMP reduction, we examined the effects of wortmannin on 10(-4) M 3-isobutyl-1-methylxantine (IBMX)-induced insulin secretion and cAMP content. In contrast to the effect on forskolin-induced secretion, wortmannin had no effect on IBMX-induced insulin secretion or cAMP content. Moreover, wortmannin had no effect on nonhydrolyzable cAMP analog-induced insulin secretion in the perfusion study. These data indicate that wortmannin induces insulin secretion by inhibiting phosphodiesterase to increase cAMP content, and suggest that PI3-kinase inhibits insulin secretion by activating phosphodiesterase to reduce cAMP content.     

Cell-penetrating peptide-conjugated lipid nanoparticles for siRNA delivery

Lipid nanoparticles (LNP) modified with cell-penetrating peptides (CPP) were prepared for the delivery of small interfering RNA (siRNA) into cells. Lipid derivatives of CPP derived from protamine were newly synthesized and used to prepare CPP-decorated LNP (CPP-LNP). Encapsulation of siRNA into CPP-LNP improved the stability of the siRNA in serum. Fluorescence-labeled siRNA formulated in CPP-LNP was efficiently internalized into B16F10 murine melanoma cells in a time-dependent manner, although that in LNP without CPP was hardly internalized into these cells. In cells transfected with siRNA in CPP-LNP, most of the siRNA was distributed in the cytoplasm of these cells and did not localize in the lysosomes. Analysis of the endocytotic pathway indicated that CPP-LNP were mainly internalized via macropinocytosis and heparan sulfate-mediated endocytosis. CPP-LNP encapsulating siRNA effectively induced RNA interference-mediated silencing of reporter genes in B16F10 cells expressing luciferase and in HT1080 human fibrosarcoma cells expressing enhanced green fluorescent protein. These data suggest that modification of LNP with the protamine-derived CPP was effective to facilitate internalization of siRNA in the cytoplasm and thereby to enhance gene silencing.     

Longitudinal distribution and microhabitat use of young Japanese eel Anguilla japonica in a small river flowing through paddy areas

Although the Japanese eel Anguilla japonica is a commercially important species, its habitat use is not well understood during its life stages in the river. In this study, we investigated the longitudinal distribution and microhabitat use of young Japanese eels (<200 mm in total length [TL], which correspond to elver and early yellow stages) using 180 quadrates (1 m × 1 m) in six stations in a small river (approximately 11.5 km long, 3.0–25.0 m wide) that flows through paddy areas in Fukushima Prefecture, Japan. No differences were observed in the TL of eels among the sampling stations. The analysis using generalized linear models showed that eel density increased as number of weirs decreased. The analysis using generalized additive models showed that water depth, current velocity, and substrate complexity were important factors determining microhabitat use. Eels used shallow habitats (<35 cm) with slow currents (5–40 cm/s) and high complex riverbeds (>0.35 in index of substrate complexity). These findings provide useful information to conserve and manage wild eels inhabiting small rivers flowing through paddy areas.     

Hydroxylations of substituted naphthalenes by Escherichia coli expressing aromatic dihydroxylating dioxygenase genes from polycyclic aromatic hydrocarbon-utilizing marine bacteria

Bioconversion experiments of various mono- or di-substituted naphthalenes such as dimethylnaphthalenes were carried out using the cells of Escherichia coli that expressed aromatic dihydroxylating dioxygenase genes (phnA1A2A3A4 and phdABCD) from polycyclic aromatic hydrocarbon-utilizing marine bacteria, Nocardioides sp. KP7 and Cycloclasticus sp. A5, respectively. We found that the former dioxygenase PhnA1A2A3A4 had broad substrate preference for these compounds and often was able to hydroxylate their methyl groups. Specifically, 1,4-dimethylnaphthalene was predominantly bioconverted into 1,4-dihydroxymethylnaphthalene.