MicroRNA-133 regulates the expression of GLUT4 by targeting KLF15 and is involved in metabolic control in cardiac myocytes

GLUT4 shows decreased levels in failing human adult hearts. We speculated that GLUT4 expression in cardiac muscle may be fine-tuned by microRNAs. Forced expression of miR-133 decreased GLUT4 expression and reduced insulin-mediated glucose uptake in cardiomyocytes. A computational miRNA target prediction algorithm showed that KLF15 is one of the targets of miR-133. It was confirmed that over-expression of miR-133 reduced the protein level of KLF15, which reduced the level of the downstream target GLUT4. Cardiac myocytes infected with lenti-decoy, in which the 3′UTR with tandem sequences complementary to miR-133 was linked to the luciferase reporter gene, had decreased miR-133 levels and increased levels of GLUT4. The expression levels of KLF15 and GLUT4 were decreased at the left ventricular hypertrophy and congestive heart failure stage in a rat model. The present results indicated that miR-133 regulates the expression of GLUT4 by targeting KLF15 and is involved in metabolic control in cardiomyocytes.     

RNA interference targeting against S100A4 suppresses cell growth and motility and induces apoptosis in human pancreatic cancer cells

S100A4 protein belongs to the S100 subfamily, which has grown to be one of the large subfamilies of the EF-hand Ca²⁺-binding proteins, and overexpression of S100A4 is suggested to associate with cell proliferation, invasion, and metastasis. We observed frequent overexpression of S100A4 in pancreatic cancer cell lines and further analyzed RNAi-mediated knockdown to address the possibility of its use as a therapeutic target for pancreatic cancer. The specific knockdown of S100A4 strongly suppressed cell growth, induced G2 arrest and eventual apoptosis, and decreased cell migration. Furthermore, microarray analyses revealed that knockdown of S100A4 induced expression of the tumor suppressor genes PRDM2 and VASH1. Our present results suggest the possibility that the inhibition of S100A4 can be utilized in antitumor applications for patients with pancreatic cancer.     

Centromeric interval of chromosome 4 derived from C57BL/6 mice accelerates type 1 diabetes in NOD.CD72 congenic mice

The nonobese diabetic (NOD) mouse is a useful model of autoimmune type 1 diabetes exhibiting many similarities to human type 1 diabetes patients including the presence of auto-reactive T cells and pancreas-specific autoantiboies. Multiple Idd loci control the development of diabetes in NOD mice. CD72, a B cell membrane-bound glycoprotein carrying a C-type lectin-like domain, is an inhibitory co-receptor of the B cell antigen receptor (BCR) that negatively regulates BCR signaling. Among four known haplotypes of mouse CD72, NOD mice carry the CD72^c haplotype, whereas most of the other inbred strains of mice carry either CD72^a or CD72^b. In this study, we generated congenic NOD.CD72^b mice that carry C57BL/6 (B6) mouse-derived centromeric chromosome 4 interval (24-45 cM) surrounding the CD72^b locus. Unexpectedly, NOD.CD72^b mice were not protected from diabetes, but rather exhibited accelerated development of both insulitis and diabetes. Our result defines novel locus or loci in the vicinity of CD72 gene that negatively control diabetes, indicating that NOD disease is under complex genetic controls of not only Idd genes but also disease-resistant genes.     

A murine model of neonatal diabetes mellitus in Glis3-deficient mice

Glis3 is a member of the Gli-similar subfamily. GLIS3 mutations in humans lead to neonatal diabetes, hypothyroidism, and cystic kidney disease. We generated Glis3-deficient mice by gene-targeting. The Glis3^−/− mice had significant increases in the basal blood sugar level during the first few days after birth. The high levels of blood sugar are attributed to a decrease in the Insulin mRNA level in the pancreas that is caused by impaired islet development and the subsequent impairment of Insulin-producing cell formation. The pancreatic phenotypes indicate that the Glis3-deficient mice are a model for GLIS3 mutation and diabetes mellitus in humans.     

Integrin-linked kinase is required for radial sorting of axons and Schwann cell remyelination in the peripheral nervous system

During development, Schwann cells (SCs) interpret different extracellular cues to regulate their migration, proliferation, and the remarkable morphological changes associated with the sorting, ensheathment, and myelination of axons. Although interactions between extracellular matrix proteins and integrins are critical to some of these processes, the downstream signaling pathways they control are still poorly understood. Integrin-linked kinase (ILK) is a focal adhesion protein that associates with multiple binding partners to link integrins to the actin cytoskeleton and is thought to participate in integrin and growth factor–mediated signaling. Using SC-specific gene ablation, we report essential functions for ILK in radial sorting of axon bundles and in remyelination in the peripheral nervous system. Our in vivo and in vitro experiments show that ILK negatively regulates Rho/Rho kinase signaling to promote SC process extension and to initiate radial sorting. ILK also facilitates axon remyelination, likely by promoting the activation of downstream molecules such as AKT/protein kinase B.     

Increased expression of CXCR4 and integrin αM in hypoxia‐preconditioned cells contributes to improved cell retention and angiogenic potency

Cell‐based angiogenesis is a promising method for the treatment of ischemic diseases, but the poor retention of implanted cells in targeted tissues is a major drawback. We tested whether hypoxic preconditioning increased retention and angiogenic potency of implanted cells in ischemic tissue. Hypoxic preconditioning of mouse peripheral blood mononuclear cells (PBMNCs) was done with 24 h of culture under 2% O₂. Normoxia‐cultured PBMNCs were used as a control. Hypoxic preconditioning increased the adhesion capacity of the PBMNCs. Moreover, the expression of integrin αM and CXCR4 was significantly higher in the hypoxia‐preconditioned PBMNCs than in the normoxia‐cultured PBMNCs. Interestingly, the expression of intercellular adhesion molecule‐1 (ICAM‐1), a ligand of integrin αM, and stromal cell‐derived factor‐1 (SDF‐1), a chemokine for CXCR4, were remarkably increased in the ischemic hindlimbs. The retention of the hypoxia‐preconditioned PBMNCs was significantly higher than that of the normoxia‐cultured PBMNCs, 3 days after their intramuscular implantation into ischemic hindlimbs. We also noted better blood flow in the ischemic hindlimbs implanted with the hypoxia‐preconditioned PBMNCs than in those implanted with the normoxia‐cultured PBMNCs, 14 days after treatment. Furthermore, antibody neutralization of integrin αM and CXCR4 abolished completely the increased cell retention and angiogenic potency of the hypoxia‐preconditioned PBMNCs after implantation into the ischemic hindlimbs. These results indicate that hypoxic preconditioning of implanted cells is a feasible method of enhancing therapeutic angiogenesis by increasing their retention. J. Cell. Physiol. 220: 508–514, 2009. © 2009 Wiley‐Liss, Inc.     

Amyloid‐β up‐regulates complement factor B in retinal pigment epithelial cells through cytokines released from recruited macrophages/microglia: Another mechanism of complement activation in age‐related macular degeneration

One of the earliest signs of age‐related macular degeneration (AMD) is the formation of drusen which are extracellular deposits beneath the retinal pigmented epithelium (RPE). To investigate the relationship between drusen and AMD, we focused on amyloid β (Aβ), a major component of drusen and also of senile plaques in the brain of Alzheimer's patients. We previously reported that Aβ was accumulated in drusen‐like structure in senescent neprilysin gene‐disrupted mice. The purpose of this study was to investigate the influence of Aβ on factor B, the main activator of the complement alternative pathway. The results showed that Aβ did not directly modulate factor B expression in RPE cells, but increased the production of monocyte chemoattractant protein‐1 (MCP‐1). Aβ also increased the production of IL‐1β and TNF‐α in macrophages/microglia, and exposure of RPE cells to IL‐1β and TNF‐α significantly up‐regulated factor B. Co‐cultures of RPE cells and macrophages/microglia in the presence of Aβ significantly increased the expression of factor B in RPE. These findings indicate that cytokines produced by macrophages/microglia that were recruited by MCP‐1 produced in RPE cells stimulated by Aβ up‐regulate factor B in RPE cells. Thus, a combined mechanism exists for Aβ‐induced for the activation of the complement alternative pathway in the subretinal space; cytokine‐induced up‐regulation of activator factor B and dysfunction of the inhibitor factor I by direct binding to Aβ as suggested in our earlier study. J. Cell. Physiol. 220: 119–128, 2009. © 2009 Wiley‐Liss, Inc.     

Molecular Characterization of a Novel Peroxidase from the Cyanobacterium Anabaena sp. Strain PCC 7120

The open reading frame alr1585 of Anabaena sp. strain PCC 7120 encodes a heme-dependent peroxidase (Anabaena peroxidase [AnaPX]) belonging to the novel DyP-type peroxidase family (EC 1.11.1.X). We cloned and heterologously expressed the active form of the enzyme in Escherichia coli. The purified enzyme was a 53-kDa tetrameric protein with a pI of 3.68, a low pH optima (pH 4.0), and an optimum reaction temperature of 35°C. Biochemical characterization revealed an iron protoporphyrin-containing heme peroxidase with a broad specificity for aromatic substrates such as guaiacol, 4-aminoantipyrine and pyrogallol. The enzyme efficiently catalyzed the decolorization of anthraquinone dyes like Reactive Blue 5, Reactive Blue 4, Reactive Blue 114, Reactive Blue 119, and Acid Blue 45 with decolorization rates of 262, 167, 491, 401, and 256 μM·min⁻¹, respectively. The apparent K_m and k_cat/K_m values for Reactive Blue 5 were 3.6 μM and 1.2 × 10⁷ M⁻¹ s⁻¹, respectively, while the apparent K_m and k_cat/K_m values for H₂O₂ were 5.8 μM and 6.6 × 10⁶ M⁻¹ s⁻¹, respectively. In contrast, the decolorization activity of AnaPX toward azo dyes was relatively low but was significantly enhanced 2- to ∼50-fold in the presence of the natural redox mediator syringaldehyde. The specificity and catalytic efficiency for hydrogen donors and synthetic dyes show the potential application of AnaPX as a useful alternative of horseradish peroxidase or fungal DyPs. To our knowledge, this study represents the only extensive report in which a bacterial DyP has been tested in the biotransformation of synthetic dyes.In textile, food, and dyestuff industries, reactive dyes such as azo and anthraquinone (AQ) and pthalocyanine-based dyes constitute one of the extensively used classes of synthetic dyes. However, it has been estimated that approximately 50% of the applied reactive dye is wasted because of hydrolysis during the dyeing process (26, 35). This results in a great effluent problem for the industries because of the recalcitrant nature of these dyes. With increased public concern and ecological awareness, in addition to stricter legislative control of wastewater discharge in recent years, there is an increased interest in various methods of dye decolorization. Dye decolorization using physicochemical processes such as coagulation, adsorption, and oxidation with ozone has proved to be effective. However, these processes are usually expensive, generate large volumes of sludge, and require the addition of environmentally hazardous chemical additives (26). There are several reports of microorganisms capable of decolorizing synthetic dyes. This has been attributed to their growth and production of enzymes such as laccase (1, 9, 40), azoreductases (3), and peroxidases, for example, lignin peroxidase (12, 25, 36), manganese peroxidase (10, 38), and versatile peroxidase (16). However, most of the synthetic dyes are xenobiotic compounds that are poorly degraded using the typical biological aerobic treatments. Furthermore, microbial anaerobic reductions of synthetic dyes are known to generate compounds such as aromatic amines that are generally more toxic than the dyes themselves (3). Therefore, for environmental safety, the use of enzymes instead of enzyme-producing microorganisms presents several advantages such as increased enzyme production, enhanced stability and/or activity, and lower costs by using recombinant DNA technology.Peroxidases are heme-containing enzymes that use hydrogen peroxide (H₂O₂) as the electron acceptor to catalyze numerous oxidative reactions. They are found widely in nature, both in prokaryotes and eukaryotes, and are largely grouped into plant and animal superfamilies. They are one of the most studied enzymes because of their inherent spectroscopic properties and potential use in both diagnostic and bioindustrial applications. In particular, their ability to degrade a wide range of substrates has recently stimulated interest in their potential application in environmental bioremediation of recalcitrant and xenobiotic wastes (10, 25, 26).Recently, a novel family of heme peroxidases characterized by broad dye decolorization activity has been identified in various fungal species such as Thanatephorus cucumeris Dec1 (18), Termitomyces albuminosus (15), Polyporaceae sp. (15), Pleurotus ostreatus (13), and Marasmius scorodonius (27). Because of their broad substrate specificity, low pH optima, lack of a conserved active site distal histidine, and structural divergence from classical plant and animal peroxidases (32), these proteins have been proposed to belong to the novel DyP peroxidase family. Over 400 proteins of prokaryotic and eukaryotic origins have been grouped in the DyP peroxidase family, Pfam 04261 (http://pfam.sanger.ac.uk/), and it is apparent from genome databases that many species possess DyP. The ability of these proteins to effectively degrade hydroxyl-free AQ and azo dyes as well as the specificity for typical peroxidase substrates illustrates their potential use in the bioremediation of wastewater contaminated with synthetic dyes. However, with the exception of a DyP from the plant pathogenic fungus T. cucumeris Dec1 (an anamorph of Rhizoctonia solani, a very common fungal plant pathogen), which has been characterized extensively (18, 28, 30-32, 34), little information is available on other members of the DyP family. In particular, studies on bacterial DyPs have been limited to only the automatically translated sequence or structural data (41, 42). Within the context of further understanding the structure-function and potential applicability of these novel types of enzymes in general, we have taken an interest in DyP-type enzymes, particularly, the less known bacterial groups.Cyanobacteria (blue-green algae) represent the most primitive, oxygenic, plant-type photosynthetic organisms and are thought to be involved in greater than 20 to 30% of the global photosynthetic primary production of biomass, accompanied by the cycling of oxygen. Anabaena sp. strain PCC 7120 is a filamentous, heterocyst-forming cyanobacterium capable of nitrogen fixation and has long been used as a model organism to study the prokaryotic genetics and physiology of cellular differentiation, pattern formation, and nitrogen fixation (14). This strain''s genome sequence is complete and annotated (17). From bioinformatics analysis of the Anabaena sp. strain PCC 7120 genome, we identified an open reading frame (ORF), alr1585, encoding a putative heme-dependent peroxidase exhibiting homology to T. cucumeris Dec1, DyP. Here, we report on the characterization of this novel bacterial DyP, designated AnaPX (for Anabaena peroxidase), from the cyanobacterium Anabaena sp. strain PCC 7120, with broad specificity for both aromatic compounds and synthetic dyes such as AQ dyes.     

Intestinal Endocellular Symbiotic Bacterium of the Macaque Louse Pedicinus obtusus: Distinct Endosymbiont Origins in Anthropoid Primate Lice and the Old World Monkey Louse

A symbiotic bacterium of the macaque louse, Pedicinus obtusus, was characterized. The symbiont constituted a gammaproteobacterial lineage distinct from the symbionts of anthropoid primate lice, localized in the midgut epithelium and the ovaries and exhibiting AT-biased genes and accelerated molecular evolution. The designation “Candidatus Puchtella pedicinophila” was proposed for it.Sucking lice (Insecta: Phthiraptera: Anoplura), ectoparasitic insects that feed exclusively on the blood of their specific mammalian hosts, are generally associated with endosymbiotic bacteria (2, 13). Recent molecular studies have demonstrated that symbiotic bacteria of sucking lice are of multiple evolutionary origins (1, 8, 10, 16).From primates, three louse genera, Pediculus, Pthirus, and Pedicinus, have been recorded. Three Pediculus and two Pthirus species are known from anthropoid primates, harboring gammaproteobacterial “Candidatus Riesia spp.” in the stomach disc (1, 16). Meanwhile, 14 Pedicinus species, whose symbiotic bacteria have not been characterized, have been recorded from Old World monkeys (3).Samples of Pedicinus obtusus were collected in 2008 at the Jigokudani Monkey Park, Nagano, Japan, where a wild population of the Japanese macaque, Macaca fuscata, is exhibited to the public under careful control. When animals were caught and inspected, samples of P. obtusus were collected from the anesthetized animals under the supervision of a veterinary doctor (permission number 19-26-24, Nagano Prefecture, Japan). Samples of P. obtusus were collected in November 2008 by courtesy of Kiyoyasu Kowaki from a subspecies of the Japanese macaque, M. fuscata yakui, that is endemic in Yakushima Island, Kagoshima, Japan. Each of the insect samples was subjected to DNA extraction, and a 1.5-kb segment of the 16S rRNA gene (5) and a 1.6-kb segment of the groEL gene (7) were amplified by PCR, cloned, and sequenced. Molecular phylogenetic analyses were performed using the programs PAUP 4.0b10 (Sinauer Associates, Sunderland, MA), RAxML version 7.0.0 (17), and MrBayes 3.1.2 (15).The 1,490-bp 16S rRNA gene sequences formed a distinct lineage in the Gammaproteobacteria, exhibiting no phylogenetic affinity to the symbionts of other louse species, including those of human lice and the chimpanzee louse (see Fig. S1 in the supplemental material). The 1,601-bp groEL gene sequences also constituted a gammaproteobacterial lineage, exhibiting no phylogenetic affinity to the symbiont of human lice (Fig. ​(Fig.1)1) These results indicated that the endosymbiotic bacterium of the Old World monkey louse evolved independently of the endosymbiotic bacteria of the anthropoid primate lice. Considering that the date of divergence of Old World monkeys and anthropoid primates has been inferred as 23 to 31 million years ago, the endosymbiotic evolution in the primate lice must have occurred within this time scale (12).Open in a separate windowFIG. 1.groEL gene sequence-based molecular phylogenetic analysis of the symbiont of P. obtusus in the Gammaproteobacteria. A maximum-likelihood tree inferred from 1,040 unambiguously aligned nucleotide sites is shown. Bayesian and neighbor-joining analyses gave essentially the same results (data not shown). Statistical support values higher than 70% are indicated at the nodes in the order of maximum-likelihood/Bayesian/neighbor-joining values. Asterisks indicate statistical support values lower than 70%. Sequence accession numbers are shown in brackets. AT contents of the sequences are also shown. The sequences from the symbionts of human and monkey lice are highlighted in boldface. P-symbiont, primary symbiont; S-symbiont, secondary symbiont.The molecular evolutionary rates of the symbiont gene sequences were analyzed with a relative rate test using the program RRTree (14). The evolutionary rates of the 16S rRNA and groEL gene sequences in the lineage of the P. obtusus symbiont were significantly higher than those in the lineages of related free-living gammaproteobacteria (see Table S1 in the supplemental material).The AT contents of the 16S rRNA and groEL gene sequences of the P. obtusus symbiont were 53.5% and 64.8%, respectively. These values were equivalent to those of obligate endosymbionts of other insects (>50% for the 16S rRNA gene and >60% for the groEL gene) and were remarkably higher than those of allied free-living gammaproteobacteria (∼45% for the 16S rRNA gene and 45 to 50% for the groEL gene) (Fig. ​(Fig.1;1; see Fig. S1 in the supplemental material).Obligate endosymbiotic bacteria that cospeciate with their host insects commonly exhibit peculiar genetic traits, including AT-biased nucleotide composition, an accelerated rate of molecular evolution, and significant genome reduction (18). The AT-biased nucleotide composition and the accelerated evolution observed with the P. obtusus symbiont (Fig. ​(Fig.1;1; see Fig. S1 and Table S1 in the supplemental material) are suggestive of a stable and intimate host-symbiont association over evolutionary time.Fluorescent in situ hybridization targeting 16S rRNA of the symbiont was performed using the Alexa Fluor 555-labeled oligonucleotide probes Al555-ML439 (Al555-5′-ATAATATCTTCTTTCCTACCGA-3′) and Al555-ML1256 (Al555-5′-GCTAATTCTTGCGAATTTGCTT-3′) as described previously (9). In first-, second-, and third-instar nymphs, the symbiont signals were detected in the posterior half of the stomach in the abdomen (Fig. ​(Fig.2A).2A). In the posterior stomach, the signals exhibited a periodical and striated pattern (Fig. ​(Fig.2B).2B). Magnified images located the symbiont signals endocellularly in the intestinal wall tissue (Fig. ​(Fig.2C).2C). In some of the third-instar nymphs, the symbiont signals were found not only in the posterior stomach but also in the ovaries (Fig. ​(Fig.2A).2A). In adult females, the symbiont signals were restricted to the lateral oviducts (Fig. ​(Fig.2A),2A), where many bacteriocytes formed a pair of symbiotic organs called ovarial ampullae (Fig. ​(Fig.2D).2D). The ovarial ampullae were located adjacent to developing oocytes in the ovarioles, where the symbiont was passed to the developing eggs (Fig. ​(Fig.2E2E).Open in a separate windowFIG. 2.Localization of the symbiont in nymphs and adults of P. obtusus. (A) General localization of the symbiont in nymphal and adult insects. The symbiont signals are seen in the posterior stomach (green arrows) and the ovarial ampullae (yellow arrows). (B) An image of the posterior stomach of a second-instar nymph. Periodical and striped regions are densely populated by the symbiont. (C) An image of the posterior stomach of a third-instar nymph. The symbiont signals are restricted to the gut epithelial cells and not detected in the stomach lumen. Bacteriocytes are intercalated with uninfected cells, forming a striated pattern. (D) An enlarged image of the ovarial ampulla, consisting of a number of uninucleated bacteriocytes. (E) An image of the ovary in an adult female, wherein developing oocytes are found in the ovarioles. Ovarial ampullae (yellow arrows) are located at the anterior tip of the lateral oviducts, where symbiont transmission to oocytes occurs (white arrow). Panel A shows epifluorescent images, while panels B to E are confocal optical sectioning images. Red and blue signals reflect the symbiont 16S rRNA and the host nuclear DNA, respectively. Abbreviations: lu, stomach lumen; oc, oocyte.These results suggested that in third-instar female nymphs of P. obtusus, the symbiont localization drastically changes, from the posterior stomach to the ovarial ampullae (Fig. ​(Fig.2A).2A). Presumably, the endocellular symbiont escapes the host cells and somehow moves to the female reproductive organ, establishing a new endocellular association and finding a way to the next host generation. Interestingly, such symbiont migrations from a symbiotic organ to the ovaries in third-instar female nymphs have been reported for the human body louse Pediculus humanus (4, 13) and the slender pigeon louse Columbicola columbae (6, 13). Here it is notable that the symbiotic bacteria of P. obtusus, P. humanus, and C. columbae are phylogenetically not related to each other (see Fig. S1 in the supplemental material). The mechanisms underpinning the symbiont migration are currently unknown. Eberle and McLean (4) suggested by a series of experiments that the ovary of the human body louse might emanate a humoral factor that attracts the symbiotic bacteria to induce the migration. Whether or not this hypothesis is true deserves future experimental studies of these louse species and their symbiotic bacteria.On the basis of these results, we propose the designation “Candidatus Puchtella pedicinophila” for the novel endosymbiont lineage. The generic name honors Otto Puchta, who identified the biological role of the human louse symbiont as the provision of B vitamins (11). The specific name indicates association with a Pedicinus monkey louse. Whether the other monkey lice harbor symbiotic bacteria allied to the symbiont of P. obtusus deserves future studies.