Nucleotide‐dependent structural fluctuations and regulation of microtubule‐binding affinity of KIF1A

Molecular motors such as kinesin regulate affinity to a rail protein during the ATP hydrolysis cycle. The regulation mechanism, however, is yet to be determined. To understand this mechanism, we investigated the structural fluctuations of the motor head of the single‐headed kinesin called KIF1A in different nucleotide states using molecular dynamics simulations of a Gō‐like model. We found that the helix at the microtubule (MT) binding site intermittently exhibits a large structural fluctuation when MT is absent. Frequency of this fluctuation changes systematically according to the nucleotide states and correlates strongly with the experimentally observed binding affinity to MT. We also showed that thermal fluctuation enhances the correlation and the interaction with the nucleotide suppresses the fluctuation of the helix . These results suggest that KIF1A regulates affinity to MT by changing the flexibility of the helix during the ATP hydrolysis process: the binding site becomes more flexible in the strong binding state than in the weak binding state. Proteins 2015; 83:809–819. © 2015 Wiley Periodicals, Inc.     

Plasma cholesterol-lowering and transient liver dysfunction in mice lacking squalene synthase in the liver

Squalene synthase (SS) catalyzes the biosynthesis of squalene, the first specific intermediate in the cholesterol biosynthetic pathway. To test the feasibility of lowering plasma cholesterol by inhibiting hepatic SS, we generated mice in which SS is specifically knocked out in the liver (L-SSKO) using Cre-loxP technology. Hepatic SS activity of L-SSKO mice was reduced by >90%. In addition, cholesterol biosynthesis in the liver slices was almost eliminated. Although the hepatic squalene contents were markedly reduced in L-SSKO mice, the hepatic contents of cholesterol and its precursors distal to squalene were indistinguishable from those of control mice, indicating the presence of sufficient centripetal flow of cholesterol and/or its precursors from the extrahepatic tissues. L-SSKO mice showed a transient liver dysfunction with moderate hepatomegaly presumably secondary to increased farnesol production. In a fed state, the plasma total cholesterol and triglyceride were significantly reduced in L-SSKO mice, primarily owing to reduced hepatic VLDL secretion. In a fasted state, the hypolipidemic effect was lost. mRNA expression of liver X receptor α target genes was reduced, while that of sterol-regulatory element binding protein 2 target genes was increased. In conclusion, liver-specific ablation of SS inhibits hepatic cholesterol biosynthesis and induces hypolipidemia without increasing significant mortality.     

Plasma carotenoid concentrations before and after supplementation with astaxanthin in middle-aged and senior subjects

A randomized, double-blind human trial was conducted to assess the effect on the plasma carotenoid concentration of 4- or 12-week astaxanthin supplementation (1 or 3 mg/d) of 20 Japanese middle-aged and senior subjects. The plasma carotenoid concentration was significantly higher after the astaxanthin supplementation than that before in both the 1 mg/d (10 subjects) and 3 mg/d (10 subjects) groups.     

Amyloid β induces adhesion of erythrocytes to endothelial cells and affects endothelial viability and functionality

It has been suggested that amyloid β-peptide (Aβ) might mediate the adhesion of erythrocytes to the endothelium which could disrupt the properties of endothelial cells. We provide evidence here that Aβ actually induced the binding of erythrocytes to endothelial cells and decreased endothelial viability, perhaps by the generation of oxidative and inflammatory stress. These changes are likely to contribute to the pathogenesis of Alzheimer's disease.     

Degeneration of hrpZ gene in Pseudomonas syringae pv. tabaci to evade tobacco defence: an arms race between tobacco and its bacterial pathogen

The HrpZ harpin of Pseudomonas syringae is known to induce a hypersensitive response (HR) in some plants. In P. syringae pv. tabaci (Pta), the harpin gene hrpZ has been spontaneously disrupted by an internal deletion in its open reading frame and a frame shift. The loss of the ability of the recombinant harpin polypeptide of Pta to induce HR despite the high sensitivity of tobacco plants to harpin led us to investigate the meaning of the disrupted hrpZ gene in the virulence of Pta 6605. The hrpZ gene from P. syringae pv. pisi was introduced into wild-type (WT) Pta. The hrpZ-complemented Pta secreted harpin into the culture medium, but failed to cause disease symptoms by both infiltration and spray inoculation. Inoculation with the hrpZ-complemented Pta induced defence responses in tobacco plants, whereas the defence responses of tobacco plants were not prominent on inoculation with WT Pta. These results indicate that an ancestor of Pta might have disrupted hrpZ by an internal deletion to evade plant defences and confer the ability to cause disease in tobacco plants.     

Mg-dechelation activity in radish cotyledons with artificial and native substrates, Mg-chlorophyllin a and chlorophyllide a.

The Mg-dechelation activity in extracts from radish (Raphanus sativus L.) cotyledons was investigated using an artificial substrate, Mg-chlorophyllin a (Chlin) and the native substrate, chlorophyllide a (Chlide). In addition to a known a small molecular weight metal-chelating substance (MCS), Mg-releasing protein (MRP) was present when Chlin was used as the substrate. However, only MCS had Mg-dechelation activity with the native substrate. To examine the possibility of the dissociation of MRP into a protein moiety and a small molecular mass compound with an activity like MCS, extraction with low and high ionic strength buffers was carried out. No evidence was obtained that MCS is a moiety of MRP, however. Inhibitor studies showed that MCS and MRP had different susceptibilities to the inhibitors, especially to the chelators tiron and EDTA when Chlin was used as the substrate. Tiron had no effect on MRP, but it severely reduced MCS activity in both substrates. The activity of MRP increased during senescence, indicating the induction of MRP, while the activity of MCS was almost unchanged. These results suggest different reaction mechanisms by independent compounds. These findings suggest that MRP and MCS are present independently, and MCS is postulated to be a substance that catalyzes the Mg-dechelation reaction in the breakdown pathway of Chl, although MCS was not induced during senescence. The properties of MRP and MCS in relation to the small molecular mass substance obtained from strawberry fruit are also discussed.     

Homeobox B3, FoxA1 and FoxA2 interactions in epithelial lung cell differentiation of the multipotent M3E3/C3 cell line

HOM/C homeobox (Hox) and forkhead box (Fox) factors are reported to be expressed in the foregut endoderm and are subsequently detected in a spatio-temporal pattern during lung development. Some of these factors were reported to influence the expression of lung marker proteins or to modulate lung development. To clarify the molecular mechanisms for generating functional lung cells from progenitor cell populations, we introduced the forkhead box factors, FoxA1 and FoxA2, and the homeobox factor, HoxB3, into the differentiation process in a multipotent hamster lung epithelial M3E3/C3 cell line. Ectopic expression of FoxA2 promoted differentiation to Clara-like cells with up-regulation of the expression of the lung marker proteins, Clara cell-specific 10-kDa protein and surfactant protein-B. In contrast, FoxA1 repressed the differentiation. HoxB3 transfection induced FoxA2 expression transiently at the pre-differentiation stage. The endogenous HoxB3 expression level decreased at later stages of Clara-like cell differentiation, and the attenuation was enhanced by FoxA2 transfection. HoxB3 is a putative upstream regulator that enhances FoxA2 expression at the pre-differentiation stage. In addition, we found that the expression of HoxA4, HoxA5, and HoxC9 increased differentially during Clara-like cell differentiation. These results suggest that HoxB3 may be a putative positive regulator of FoxA2 expression at the pre-differentiation stage, and those interactions of Fox factors and Hox factors could participate in Clara cell differentiation.     

Structural and functional analysis of pancreatic islets preserved by pioglitazone in db/db mice

To evaluate preventive effects of pioglitazone on pancreatic beta-cell damage in C57BL/KsJ db/db mice, an obese diabetic animal model, the pancreatic islets were compared morphologically between pioglitazone-treated (100 mg/kg daily po) and untreated db/db mice (n = 7 for each) after a 12-wk intervention (6-18 wk of age). The fasting blood glucose level was significantly improved by the treatment with pioglitazone (260 +/- 12 vs. 554 +/- 62 mg/dl, P < 0.05). The islet mass in the pancreas was significantly greater in pioglitazone-treated mice than in untreated mice (10.2 +/- 1.1 vs. 4.6 +/- 0.2 mg, P < 0.01). Subsequently, biochemical and physiological analyses of the beta-cell function were employed using pioglitazone-treated and untreated db/db mice (n = 6 for each) and pioglitazone-treated and untreated db/+ mice (n = 6 for each). After 2 wk of treatment (10-12 wk of age), the plasma levels of triglyceride and free fatty acid were significantly decreased, whereas the plasma adiponectin level increased significantly compared with the untreated group (65.2 +/- 18.0 vs. 18.3 +/- 1.3 microg/ml, P < 0.05). Pioglitazone significantly reduced the triglyceride content in the islets (43.3 +/- 3.6 vs. 65.6 +/- 7.6 ng/islet, P < 0.05) with improved glucose-stimulated insulin secretion. Pioglitazone showed no significant effects on the biochemical and physiological parameters in db/+ mice. The present study first demonstrated that pioglitazone prevents beta-cell damage in an early stage of the disease progression in db/db mice morphologically and physiologically. Our results suggest that pioglitazone improves glucolipotoxicity by increasing insulin sensitivity and reducing fat accumulation in the pancreatic islets.     

Morphological analysis of ghrelin and its receptor distribution in the rat pancreas

Ghrelin, a novel peptide isolated from stomach tissue of rats and humans, has been identified as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In addition to its secretion from the stomach, ghrelin is also expressed in the hypothalamic arcuate nucleus, intestine, kidney, placenta, and pancreas. GHS-R mRNA, on the other hand, is expressed in the hypothalamus, pituitary, heart, lung, liver, pancreas, stomach, intestine, and adipose tissue. Ghrelin is considered to have important roles in feeding regulation and energy metabolism as well as in the release of growth hormone (GH). Recent physiological experiments on the pancreas have shown that ghrelin regulates insulin secretion. However, sites of action of ghrelin in the pancreas are yet to be identified. In this study, to gain insight into the role of ghrelin in rat pancreatic islets, we used immunohistochemistry to determine the localization of ghrelin and GHS-R in islet cells. Double fluorescence immunohistochemistry revealed that weak GHS-R-like immunoreactivity was found in B cells containing insulin. GHS-R immunoreactivity overlapped that of glucagon-like immunoreactive cells. Moreover, both ghrelin and GHS-R-like immunoreactivities were detected mostly in the same cells in the periphery of the islets of Langerhans. These observations suggest that ghrelin is synthesized and secreted from A cells, and acts back on A cells in an autocrine and/or paracrine manner. In addition, ghrelin may act on B cells via GHS-R to regulate insulin secretion.