Expression and purification of human (pro)renin receptor in insect cells using baculovirus expression system

The renin-angiotensin (RA) system is important for the regulation of blood pressure and electrolyte balance, and renin is the rate-limiting enzyme in this system. The recent discovery of (pro)renin receptor (PRR) has reinforced the functional role of the RA system. PRR non-proteolytically activates prorenin and its role has attracted the attention of researchers towards the RA system. However, there is insufficient information on the biochemical structure and biological functioning of PRR due to the difficulty of measuring PRR expression. In this work, human PRR (hPRR) with intact transmembrane and C-terminal domain (hPRR-wTM) and PRR without this domain (hPRR-w/oTM) were expressed in insect cells using baculovirus expression system (BES). Both hPRR-wTM and hPRR-w/oTM were fused with FLAG peptide by its N-terminus. Most of the hPRR-wTM was expressed in cell fraction and hPRR-w/oTM was secreted into the culture medium. hPRR-wTM was solubilized from the membrane fraction of recombinant baculovirus-infected cells by various detergents, suggesting that hPRR-wTM might be a transmembrane protein. hPRR-wTM was purified from the solubilized fraction using anti-FLAG M2 antibody agarose. Binding of purified hPRR-wTM to renin immobilized onto sensor chips was directly proportional to the hPRR-wTM concentration. Approximately 225 μg of functional hPRR-wTM was purified from 80 ml of baculovirus-infected cell culture. Scale-up of this system will lead to mass production and crystallization of hPRR-wTM and determination of its biochemical structure and biological function.     

Single-cell gene profiling defines differential progenitor subclasses in mammalian neurogenesis

Cellular diversity of the brain is largely attributed to the spatial and temporal heterogeneity of progenitor cells. In mammalian cerebral development, it has been difficult to determine how heterogeneous the neural progenitor cells are, owing to dynamic changes in their nuclear position and gene expression. To address this issue, we systematically analyzed the cDNA profiles of a large number of single progenitor cells at the mid-embryonic stage in mouse. By cluster analysis and in situ hybridization, we have identified a set of genes that distinguishes between the apical and basal progenitors. Despite their relatively homogeneous global gene expression profiles, the apical progenitors exhibit highly variable expression patterns of Notch signaling components, raising the possibility that this causes the heterogeneous division patterns of these cells. Furthermore, we successfully captured the nascent state of basal progenitor cells. These cells are generated shortly after birth from the division of the apical progenitors, and show strong expression of the major Notch ligand delta-like 1, which soon fades away as the cells migrate in the ventricular zone. We also demonstrated that attenuation of Notch signals immediately induces differentiation of apical progenitors into nascent basal progenitors. Thus, a Notch-dependent feedback loop is likely to be in operation to maintain both progenitor populations.     

COPI–TRAPPII activates Rab18 and regulates its lipid droplet association

The transport protein particle (TRAPP) was initially identified as a vesicle tethering factor in yeast and as a guanine nucleotide exchange factor (GEF) for Ypt1/Rab1. In mammals, structures and functions of various TRAPP complexes are beginning to be understood. We found that mammalian TRAPPII was a GEF for both Rab18 and Rab1. Inactivation of TRAPPII‐specific subunits by various methods including siRNA depletion and CRISPR–Cas9‐mediated deletion reduced lipolysis and resulted in aberrantly large lipid droplets. Recruitment of Rab18 onto lipid droplet (LD) surface was defective in TRAPPII‐deleted cells, but the localization of Rab1 on Golgi was not affected. COPI regulates LD homeostasis. We found that the previously documented interaction between TRAPPII and COPI was also required for the recruitment of Rab18 to the LD. We hypothesize that the interaction between COPI and TRAPPII helps bring TRAPPII onto LD surface, and TRAPPII, in turn, activates Rab18 and recruits it on the LD surface to facilitate its functions in LD homeostasis.     

Genomics approach to abscisic acid- and gibberellin-responsive genes in rice.

We used an 8987-EST collection to construct a cDNA microarray system with various genomics information (full-length cDNA, expression profile, high accuracy genome sequence, phenotype, genetic map, and physical map) in rice. This array was used as a probe to hybridize target RNAs prepared from normally grown callus of rice and from callus treated for 6 hr or 3 days with the hormones abscisic acid (ABA) or gibberellin (GA). We identified 509 clones, including many clones that had never been annotated as ABA-or GA-responsive. These genes included not only ABA- or GA-responsive genes but also genes responsive to other physiological conditions such as pathogen infection, heat shock, and metal ion stress. Comparison of ABA- and GA-responsive genes revealed antagonistic regulation for these genes by both hormones except for one defense-related gene, thionin. The gene for thionin was up-regulated by both hormone treatments for 3 days. The upstream regions of all the genes that were regulated by both hormones had cis-elements for ABA and GA response. We performed a clustering analysis of genes regulated by both hormones and various expression profiles that showed three notable clusters (seed tissues, low temperature and sugar starvation, and thionin-gene related). A comparison of the cis-elements for hormone response genes between rice and Arabidopsis thaliana, we identified cis-elements for dehydration-stress response or for expression of amylase gene as Arabidopsis gene-specific or rice gene-specific, respectively.     

A model of cancer stem cells derived from mouse induced pluripotent stem cells

Cancer stem cells (CSCs) are capable of continuous proliferation and self-renewal and are proposed to play significant roles in oncogenesis, tumor growth, metastasis and cancer recurrence. CSCs are considered derived from normal stem cells affected by the tumor microenvironment although the mechanism of development is not clear yet. In 2007, Yamanaka's group succeeded in generating Nanog mouse induced pluripotent stem (miPS) cells, in which green fluorescent protein (GFP) has been inserted into the 5'-untranslated region of the Nanog gene. Usually, iPS cells, just like embryonic stem cells, are considered to be induced into progenitor cells, which differentiate into various normal phenotypes depending on the normal niche. We hypothesized that CSCs could be derived from Nanog miPS cells in the conditioned culture medium of cancer cell lines, which is a mimic of carcinoma microenvironment. As a result, the Nanog miPS cells treated with the conditioned medium of mouse Lewis lung carcinoma acquired characteristics of CSCs, in that they formed spheroids expressing GFP in suspension culture, and had a high tumorigenicity in Balb/c nude mice exhibiting angiogenesis in vivo. In addition, these iPS-derived CSCs had a capacity of self-renewal and expressed the marker genes, Nanog, Rex1, Eras, Esg1 and Cripto, associated with stem cell properties and an undifferentiated state. Thus we concluded that a model of CSCs was originally developed from miPS cells and proposed the conditioned culture medium of cancer cell lines might perform as niche for producing CSCs. The model of CSCs and the procedure of their establishment will help study the genetic alterations and the secreted factors in the tumor microenvironment which convert miPS cells to CSCs. Furthermore, the identification of potentially bona fide markers of CSCs, which will help the development of novel anti-cancer therapies, might be possible though the CSC model.     

Involvement of ERK Phosphorylation of Trigeminal Spinal Subnucleus Caudalis Neurons in Thermal Hypersensitivity in Rats with Infraorbital Nerve Injury

To evaluate the involvement of the mitogen-activated protein kinase (MAPK) cascade in orofacial neuropathic pain mechanisms, this study assessed nocifensive behavior evoked by mechanical or thermal stimulation of the whisker pad skin, phosphorylation of extracellular signal-regulated kinase (ERK) in trigeminal spinal subnucleus caudalis (Vc) neurons, and Vc neuronal responses to mechanical or thermal stimulation of the whisker pad skin in rats with the chronic constriction nerve injury of the infraorbital nerve (ION-CCI). The mechanical and thermal nocifensive behavior was significantly enhanced on the side ipsilateral to the ION-CCI compared to the contralateral whisker pad or sham rats. ION-CCI rats had an increased number of phosphorylated ERK immunoreactive (pERK-IR) cells which also manifested NeuN-IR but not GFAP-IR and Iba1-IR, and were significantly more in ION-CCI rats compared with sham rats following noxious but not non-noxious mechanical stimulation. After intrathecal administration of the MEK1 inhibitor PD98059 in ION-CCI rats, the number of pERK-IR cells after noxious stimulation and the enhanced thermal nocifensive behavior but not the mechanical nocifensive behavior were significantly reduced in ION-CCI rats. The enhanced background activities, afterdischarges and responses of wide dynamic range neurons to noxious mechanical and thermal stimulation in ION-CCI rats were significantly depressed following i.t. administration of PD98059, whereas responses to non-noxious mechanical and thermal stimulation were not altered. The present findings suggest that pERK-IR neurons in the Vc play a pivotal role in the development of thermal hypersensitivity in the face following trigeminal nerve injury.     

AXENIC TISSUE CULTURE AND MORPHOGENESIS IN PORPHYRA YEZOENSIS (BANGIALES, RHODOPHYTA)

To better understand developmental phenomena in macroalgal tissue culture, we examined the morphogenesis of Porphyra yezoensis Ueda (strain TU-1) cultured aseptically in defined synthetic media . Generally, the filamentous thalli (sporophyte; conchocelis phase) of P. yezoensis were densely tufted with uniseriate filaments. The foliose thalli (gametophyte) were monolayered. In this study, axenic filamentous thalli retained their characteristic morphogenesis; there were no obvious differences between morphogenetic traits in unialgal and axenic conditions. However, conchospores, which might have developed into the foliose form under unialgal conditions, germinated into calluslike masses under axenic conditions. Most of the gametophytes gradually lost their typical morphogenesis after the first longitudinal cell division. Some of the calluslike masses developed rhizoidlike structures in several places or along the entire mass. Therefore, we concluded that P. yezoensis, in axenic cultures, loses its typical morphogenesis only during the gametophytic phase. The axenic tissue culture of Porphyra established in this study is a promising assay system for the identification of growth and morphogenetic factors.     

Gastroprotective effect of leukotriene receptor blocker montelukast in alendronat-induced lesions of the rat gastric mucosa

Alendronate causes serious gastrointestinal adverse effects. We aimed to investigate if montelukast, a leukotriene receptor antagonist, is protective against this damage. Rats were administered 20 mg/kg alendronate by gavage for 4 days, either alone or following treatment with montelukast (10 mg/kg). On the last day, following drug administration, pilor ligation was performed and 2 h later, rats were killed and stomach, liver and kidney tissues were removed. Gastric acidity, gastric tissue ulcer index values and malondialdehyde (MDA); an end product of lipid peroxidation, and glutathione (GSH) levels; a key antioxidant, as well as myeloperoxidase (MPO) activity; an indirect marker of tissue neutrophil infiltration were determined, and the histologic appearance of the stomach, liver and kidney tissues were studied. Chronic oral administration of alendronate induced significant gastric damage, increasing myeloperoxidase activity and lipid peroxidation, while tissue glutathione levels decreased. Similarly, in the alendronate group MDA levels and MPO activities of liver and kidney tissues were increased and GSH levels were decreased. Treatment with montelukast prevented the damage as well as the changes in biochemical parameters in all tissues studied. Findings of the present study suggest that alendronate is a local irritant that causes inflammation through neutrophil infiltration and oxidative damage in tissues, and that montelukast is protective against this damage by its anti-inflammatory effect.     

A cryptic Rab1-binding site in the p115 tethering protein

Small GTPases and coiled-coil proteins of the golgin family help to tether COPI vesicles to Golgi membranes. At the cis-side of the Golgi, the Rab1 GTPase binds directly to each of three coiled-coil proteins: p115, GM130, and as now shown, Giantin. Rab1 binds to a coiled-coil region within the tail domain of p115 and this binding is inhibited by the C-terminal, acidic domain of p115. Furthermore, GM130 and Giantin bind to the acidic domain of p115 and stimulate p115 binding to Rab1, suggesting that p115 binding to Rab1 is regulated. Regulation of this interaction by proteins such as GM130 and Giantin may control the membrane recruitment of p115 by Rab1.     

Mapping the functional domains of the Golgi stacking factor GRASP65

The Golgi reassembly stacking protein (GRASP) family has been implicated in the stacking of Golgi cisternae and the regulation of Golgi disassembly/reassembly during mitosis in mammalian cells. GRASP65 is a dimer that can directly link adjacent surfaces through trans-oligomerization in a mitotically regulated manner. Here we show that the N-terminal GRASP domain (amino acids 1-201) is both necessary and sufficient for dimerization and trans-oligomerization but is not mitotically regulated. The C-terminal serine/proline-rich domain (amino acids 202-446) cannot dimerize nor can it link adjacent surfaces. It does, however, confer mitotic regulation on the GRASP domain through multiple sites phosphorylated by the mitotic kinases, cdc2/B1, and the polo-like kinase. Transient expression corroborated these results by showing that the GRASP domain alone inhibited mitotic fragmentation of the Golgi apparatus.