Direct monitoring of in vivo ER stress during the development of insulin resistance with ER stress-activated indicator transgenic mice

Type 2 diabetes is one of the most prevalent and serious metabolic diseases in the world, and insulin resistance and pancreatic β-cell dysfunction are the hallmarks of the disease. It has been suggested that endoplasmic reticulum (ER) stress is provoked under diabetic conditions and is possibly involved in the development of insulin resistance. In this study, using ER stress-activated indicator (ERAI) transgenic mice which express green fluorescent protein (GFP) under ER stress conditions, we directly monitored in vivo ER stress in various insulin target tissues such as liver, fat, and muscle in diabetic mice with insulin resistance induced by high fat and high sucrose (HF/HS) diet treatment. In the liver of the ERAI transgenic mice, ERAI fluorescence activity was clearly observed as early as after 4 weeks of HF/HS diet treatment, whereas it was not detected at all in the fat and muscle even after 12 weeks of HF/HS diet treatment. These results suggest that induction of ER stress is associated with the development of insulin resistance and that ER stress in the liver may facilitate the development of insulin resistance in the whole body. This is the first report to directly monitor in vivo ER stress in various insulin target tissues during the development of insulin resistance. In addition, our present results suggest that ERAI transgenic mice are very useful for evaluating in vivo ER stress, especially in the liver, during the development of insulin resistance.     

Domain architecture of the atypical Arf-family GTPase Arl13b involved in cilia formation

Arl13b is an atypical Arf/Arl-family GTPase consisting of an extending large C-terminal region (C domain) and Arf-homologous GTP-binding motifs in the N terminus (N domain). Although Arl13b appears to be involved in cilia formation, its precise function and roles of the domains remain unknown. Here, we show the unique domain architecture of Arl13b by analyzing the relationship between its biochemical properties and cilia formation. Arl13b binds guanine nucleotides and specifically localizes to cilia. The ciliary localization of Arl13b requires both N and C domains but is independent of its guanine nucleotide-binding ability. Arl13b is capable of self-associating via N domain, and overexpression of N domain inhibits not only cilia formation but also the maintenance of pre-generated cilia. These findings suggest that N and C domains of Arl13b cooperatively regulate its ciliary localization and that N domain-dependent self-association of Arl13b may be important for its function in cilia biogenesis.     

Mechanisms for the proliferation of eosinophilic leukemia cells by FIP1L1-PDGFRalpha

The constitutively activated tyrosine kinase Fip1-like 1 (FIP1L1)-platelet-derived growth factor receptor α (PDGFRα) causes eosinophilic leukemia EoL-1 cells to proliferate. Recently, we demonstrated that histone deacetylase inhibitors suppressed this proliferation and induced the differentiation of EoL-1 cells into eosinophils in parallel with a decrease in the level of FIP1L1-PDGFRα. In this study, we analyzed the mechanism by which FIP1L1-PDGFRα induces the proliferation and whether the suppression of cell proliferation triggers the differentiation into eosinophils. The FIP1L1-PDGFRα inhibitor imatinib inhibited the proliferation of EoL-1 cells and decreased the level of the oncoprotein c-Myc as well as the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK). The proliferation of EoL-1 cells and expression of c-Myc were also inhibited by the MEK inhibitor U0126 and JNK inhibitor SP600125. The expression of the eosinophilic differentiation marker CCR3 was not induced by imatinib. These findings suggest that FIP1L1-PDGFRα induces the proliferation of EoL-1 cells through the induction of c-Myc expression via ERK and JNK signaling pathways, but is not involved in the inhibition of differentiation toward mature eosinophils.     

Changes in cell morphology and cytoskeletal organization are induced by human mitotic checkpoint gene, Bub1

The mitotic spindle checkpoint prevents the onset of anaphase and subsequent cell division until chromosomes are properly aligned on a bipolar spindle. Thus, it regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. The budding uninhibited by benzimidazole (Bub1) is a key component of mitotic checkpoint. Bub1 encodes a serine/threonine kinase required for mitotic spindle checkpoint function. The regulation of cell morphology in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). Here we show that Bub1 directly affects the structural integrity of IFs. Constitutive expression of Bub1 caused disappearance of filamentous vimentin, a type III IF, and consequently changed cell morphology. Expression of kinase domain—deleted Bub1 induced neither morphological change nor disappearance of vimentin. These observations suggest that Bub1 not only regulates the cell cycle, but also may be involved in the cytoskeletal control in interphase cells.     

Synergistic action of gastrin and ghrelin on gastric acid secretion in rats

Gastrin and ghrelin are secreted from G cells and X/A-like cells in the stomach, respectively, and respective hormones stimulate gastric acid secretion by acting through histamine and the vagus nerve. In this study, we examined the relationship between gastrin, ghrelin and gastric acid secretion in rats. Intravenous (iv) administration of 3 and 10 nmol of gastrin induced transient increases of ghrelin levels within 10 min in a dose-dependent manner. Double immunostaining for ghrelin and gastrin receptor revealed that a proportion of ghrelin cells possess gastrin receptors. Although (iv) administration of gastrin or ghrelin induced significant gastric acid secretion, simultaneous treatment with both hormones resulted in a synergistic, rather than additive, increase of gastric acid secretion. This synergistic increase was not observed in vagotomized rats.These results suggest that gastrin may directly stimulate ghrelin release from the stomach, and that both hormones may increase gastric acid secretion synergistically.     

Expression profile analysis of aorta-gonad-mesonephros region-derived stromal cells reveals genes that regulate hematopoiesis

The aorta-gonad-mesonephros (AGM) region is involved in the generation and maintenance of the first definitive hematopoietic stem cells (HSCs). A mouse AGM-derived cell line, AGM-S3, was shown to support the development of HSCs. To elucidate the molecular mechanisms regulating early hematopoiesis, we obtained subclones from AGM-S3, one of which was hematopoiesis supportive (S3-A9) and the other one of which was non-supportive (S3-A7), and we analyzed their gene expression profiles by gene chip analysis. In the present study, we found that Glypican-1 (GPC1) was highly expressed in the supportive subclone AGM-S3-A9. Over-expression of GPC1 in non-supportive cells led to the proliferation of progenitor cells in human cord blood when cocultured with the transfected-stromal cells. Thus, GPC1 may have an important role in the establishment of a microenvironment that supports early events in hematopoiesis.     

Amyloid nucleation triggered by agitation of beta2-microglobulin under acidic and neutral pH conditions

Amyloid nucleation through agitation was studied with beta2-microglobulin, which is responsible for dialysis-related amyloidosis, in the presence of salt under acid and neutral pH conditions. First, the aggregation of beta2-microglobulin in NaCl solutions was achieved by mildly agitating for 24 h at 37 degrees C protein solutions in three different states: acid-unfolded, salt-induced protofibrillar, and native. The formation of aggregates was confirmed by an increase in light scattering intensity of the solutions. Then, the aggregated samples were incubated without agitation at 37 degrees C for up to 25-45 days. The structural changes in the aggregated state during the incubation period were examined by means of fluorescence spectroscopy with thioflavin T, circular dichroism spectroscopy, and electron microscopy. The results revealed that all the samples in the different states produced a mature amyloid nucleus upon agitation, after which the fibrils elongated without any detectable lag phase during the incubation, with the acid-unfolded protein better suited to undergoing the structural rearrangements necessary to form amyloid fibrils than the more structured forms. The amount of aggregate including the amyloid nucleus produced by agitation from the native conformation at neutral pH was estimated to be about 9% of all the protein by an analysis using ultracentrifugation. Additionally, amyloid nucleation by agitation was similarly achieved for a different protein, hen egg-white lysozyme, in 0.5 M NaCl solution at neutral pH. Taken together, the agitation-treated aggregates of both proteins have a high propensity to produce an amyloid nucleus even at neutral pH, providing evidence that the aggregation pathway involves amyloid nucleation under entirely native conditions.     

Monooxygenase activity of Octopus vulgaris hemocyanin

Octopus vulgaris hemocyanin ( Ov-Hc) and one of its minimal functional units ( Ov-g) have been purified, and their spectroscopic features and monooxygenase (phenolase) activity have been examined in detail. The oxy forms of both Ov-Hc and Ov-g are stable in 0.5 M borate buffer (pH 9.0) even in the presence of a high concentration of urea at 25 degrees C; approximately 90 and approximately 75% of the (mu-eta (2):eta (2)-peroxo)dicopper(II) species of Ov-Hc and Ov-g, respectively, remained unchanged after argon (Ar) gas flushing of the sample solutions for 1 h. The catalytic activity of Ov-g in the oxygenation reaction (multiturnover reaction) of 4-methylphenol ( p-cresol) to 4-methyl-1,2-dihydroxybenzene (4-methylcatechol) was higher than that of Ov-Hc, and its catalytic activity was further accelerated by the addition of urea. Kinetic deuterium isotope effect analysis and Hammett analysis using a series of phenol derivatives under anaerobic conditions (single-turnover reaction) have indicated that the monooxygenation reaction of phenols to catechols by the peroxo species of oxyhemocyanin proceeds via electrophilic aromatic substitution mechanism as in the case of tyrosinase. The effect of urea on the redox functions of oxyhemocyanin is discussed on the basis of the spectroscopic analysis and reactivity studies.     

Enhanced Levels of the Aroma and Flavor Compound S-Linalool by Metabolic Engineering of the Terpenoid Pathway in Tomato Fruits

The aromas of fruits, vegetables, and flowers are mixtures of volatile metabolites, often present in parts per billion levels or less. We show here that tomato (Lycopersicon esculentum Mill.) plants transgenic for a heterologous Clarkia breweri S-linalool synthase (LIS) gene, under the control of the tomato late-ripening-specific E8 promoter, synthesize and accumulate S-linalool and 8-hydroxylinalool in ripening fruits. Apart from the difference in volatiles, no other phenotypic alterations were noted, including the levels of other terpenoids such as gamma- and alpha-tocopherols, lycopene, beta-carotene, and lutein. Our studies indicate that it is possible to enhance the levels of monoterpenes in ripening fruits by metabolic engineering.     

Gene cloning and characterization of alanine racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei.

Alanine racemase genes (alr) from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei were cloned and expressed in Escherichia coli JM109. All genes encoded a polypeptide of 359 amino acids, and showed more than 99% sequence identities with each other. In particular, the S. dysenteriae alr was identical with the S. flexneri alr. Differences in the amino acid sequences between the four Shigella enzymes were only two residues: Gly138 in S. dysenteriae and S. flexneri (Glu138 in the other) and Ile225 in S. sonnei (Thr225 in the other). The S. boydii enzyme was identical with the E. coli K12 alr enzyme. Each Shigella alr enzyme purified to homogeneity has an apparent molecular mass about 43,000 by SDS-gel electrophoresis, and about 46,000 by gel filtration. However, all enzymes showed an apparent molecular mass about 60,000 by gel filtration in the presence of a substrate, 0.1 M l-alanine. These results suggest that the Shigella alr enzymes having an ordinary monomeric structure interact with other monomer in the presence of the substrate. The enzymes were almost identical in the enzymological properties, and showed lower catalytic activities (about 210 units/mg) than those of homodimeric alanine racemases reported.