KRAS-induced actin-interacting protein is required for the proper localization of inositol 1,4,5-trisphosphate receptor in the epithelial cells

Three inositol 1,4,5-trisphosphate receptor (IP₃R) subtypes are differentially expressed among tissues and function as the Ca²⁺ release channel on specialized endoplasmic reticulum (ER) membranes. The proper subcellular localization of IP₃R is crucial for its proper function, but this molecular mechanism is unclear. KRAS-induced actin-interacting protein (KRAP) was originally identified as a cancer-related molecule, and is involved in the regulation of whole-body energy homeostasis and pancreatic exocrine system. We herein identified IP₃R as an associated molecule with KRAP in vivo, and the association was validated by the co-immunoprecipitation and confocal immunostaining studies in mouse tissues including liver and pancreas. The association of KRAP with IP₃R was also observed in the human epithelial cell lines including HCT116, HeLa and HEK293 cells. Intriguingly, KRAP interacts with distinct subtypes of IP₃R in a tissue-dependent manner, i.e. IP₃R1 and IP₃R2 in the liver and IP₃R2 and IP₃R3 in the pancreas. The NH₂-terminal amino acid residues 1–610 of IP₃R are critical for the association with KRAP and KRAP–IP₃R complex resides in a specialized ER but not a typical reticular ER. Furthermore, the localization of particular IP₃R subtypes in tissues from KRAP-deficient mice is obviously disturbed, i.e. IP₃R1 and IP₃R2 in the liver and IP₃R2 and IP₃R3 in the pancreas. These findings demonstrate that KRAP physically associates with IP₃R and regulates the proper localization of IP₃R in the epithelial cells in vivo and cultured cells, and might shed light on the Ca²⁺ signaling underlying physiological cellular programs, cancer development and metabolism-related diseases.     

Induction of antigen-specific immunity by pH-sensitive carbonate apatite as a potent vaccine carrier

The ability of carbonate apatite (CO₃Ap) to enhance antigen-specific immunity was examined in vitro and in vivo to investigate its utility as a vaccine carrier. Murine bone marrow-derived dendritic cells took up ovalbumin (OVA) containing CO₃Ap more effectively than free OVA. Interestingly, mice immunized with OVA-containing CO₃Ap produced OVA-specific antibodies more effectively than mice immunized with free OVA. Furthermore, immunization of C57BL/6 mice with OVA-containing CO₃Ap induced the proliferation and antigen-specific production of IFN-γ by splenocytes more strongly than immunization with free OVA. Moreover, no significant differences were detected in the induction of delayed-type hypersensitivity responses, an immune reaction involving an antigen-specific, cell-mediated immune response between OVA-containing CO₃Ap and OVA-containing alumina salt (Alum), suggesting that CO₃Ap induced cell-mediated immune response to the same degree as Alum, which is commonly used for clinical applications. This study is the first to demonstrate the induction of antigen-specific immune responses in vivo by CO₃Ap.     

Hes1 regulates the number and anterior-posterior patterning of mesencephalic dopaminergic neurons at the mid/hindbrain boundary (isthmus)

The lack of the Hes1 gene leads to the failure of cranial neurulation due to the premature onset of neural differentiation. Hes1 homozygous null mutant mice displayed a neural tube closure defect, and exencephaly was induced at the mid/hindbrain boundary. In the mutant mesencephalon, the roof plate was not formed and therefore the ventricular zone showing cell proliferation was displaced to the brain surface. Furthermore, the telencephalon and ventral diencephalon were defective. Despite the severe defects of neurogenesis in null mutants, the mesencephalic dopaminergic (mesDA) neurons were specified at the midline of the ventral mesencephalon in close proximity to two important signal centers — floor plate and mid/hindbrain boundary (i.e., the isthmic organizer). Using mesDA neuronal markers, tyrosine hydroxylase (TH) and Pitx3, the development of mesDA neurons was studied in Hes1 null mice and compared with that in the wild type. At early stages, between embryonic day (E) 11.5 and E12.5, mesDA neurons were more numerous in null mutants than in the wild type. From E13.5 onward, however, the cell number and fiber density of mesDA neurons were decreased in the mutants. Their distribution pattern was also different from that of the wild type. In particular, mesDA neurons grew dorsally and invaded the rostral hindbrain. 5-HT neurons were also ectopically located in the mutant midbrain. Thus, the loss of Hes1 resulted in disturbances in the inductive and repulsive activities of the isthmic organizer. It is proposed that Hes1 plays a role in regulating the location and density of mesDA neurons.     

Inhibitors of the Influenza A Virus M2 Proton Channel Discovered Using a High-Throughput Yeast Growth Restoration Assay

The M2 proton channel of the influenza A virus is the target of the anti-influenza drugs amantadine and rimantadine. The effectiveness of these drugs has been dramatically limited by the rapid spread of drug resistant mutations, mainly at sites S31N, V27A and L26F in the pore of the channel. Despite progress in designing inhibitors of V27A and L26F M2, there are currently no drugs targeting these mutated channels in clinical trials. Progress in developing new drugs has been hampered by the lack of a robust assay with sufficient throughput for discovery of new active chemotypes among chemical libraries and sufficient sensitivity to provide the SAR data essential for their improvement and development as drugs. In this study we adapted a yeast growth restoration assay, in which expression of the M2 channel inhibits yeast growth and exposure to an M2 channel inhibitor restores growth, into a robust and sensitive high-throughput screen for M2 channel inhibitors. A screen of over 250,000 pure chemicals and semi-purified fractions from natural extracts identified 21 active compounds comprising amantadine, rimantadine, 13 related adamantanes and 6 non-adamantanes. Of the non-adamantanes, hexamethylene amiloride and a triazine derivative represented new M2 inhibitory chemotypes that also showed antiviral activity in a plaque reduction assay. Of particular interest is the fact that the triazine derivative was not sufficiently potent for detection as an inhibitor in the traditional two electrode voltage clamp assay for M2 channel activity, but its discovery in the yeast assay led to testing of analogues of which one was as potent as amantadine.     

Isolation and Characterization of Antimicrobial Compounds in Plant Extracts against Multidrug-Resistant Acinetobacter baumannii

The number of fully active antibiotic options that treat nosocomial infections due to multidrug-resistant Acinetobacter baumannii (A. baumannii) is extremely limited. Magnolia officinalis, Mahonia bealei, Rabdosia rubescens, Rosa rugosa, Rubus chingii, Scutellaria baicalensis, and Terminalia chebula plant extracts were previously shown to have growth inhibitory activity against a multidrug-resistant clinical strain of A. baumannii. In this study, the compounds responsible for their antimicrobial activity were identified by fractionating each plant extract using high performance liquid chromatography, and determining the antimicrobial activity of each fraction against A. baumannii. The chemical structures of the fractions inhibiting >40% of the bacterial growth were elucidated by liquid chromatography/mass spectrometry analysis and nuclear magnetic resonance spectroscopy. The six most active compounds were identified as: ellagic acid in Rosa rugosa; norwogonin in Scutellaria baicalensis; and chebulagic acid, chebulinic acid, corilagin, and terchebulin in Terminalia chebula. The most potent compound was identified as norwogonin with a minimum inhibitory concentration of 128 µg/mL, and minimum bactericidal concentration of 256 µg/mL against clinically relevant strains of A. baumannii. Combination studies of norwogonin with ten anti-Gram negative bacterial agents demonstrated that norwogonin did not enhance the antimicrobial activity of the synthetic antibiotics chosen for this study. In conclusion, of all identified antimicrobial compounds, norwogonin was the most potent against multidrug-resistant A. baumannii strains. Further studies are warranted to ascertain the prophylactic and therapeutic potential of norwogonin for infections due to multidrug-resistant A. baumannii.     

PTIP Associated protein 1, PA1, Is an Independent Prognostic Factor for Lymphnode Negative Breast Cancer

Pax transactivation domain interacting protein (PTIP) associated protein 1, PA1, was a newly found protein participating in the modulation of transactivity of nuclear receptor super family members such as estrogen receptor (ER), androgen receptor (AR) and glucocorticoid receptor (GR). Breast cancer is one of the most life threatening diseases for women and has tight association with estrogen and ER. This study was performed to understand the function of PA1 in breast cancer. The expression of PA1 had been evaluated in a total of 344 primary invasive breast cancer samples and examined the relationship with clinical output, relapse free survival (RFS), breast cancer-specific survival (BCSS). PA1 expression was observed in both nucleus and cytoplasm, however, appeared mainly in nuclear. PA1 nuclear expression was correlated with postmenopausal (P = 0.0097), smaller tumor size (P = 0.0025), negative Ki67 (P = 0.02), positive AR (P = 0.049) and positive ERβ (P = 0.0020). Kaplan–Meier analysis demonstrated PA1 nuclear positive cases seemed to have a longer survival than negative ones for RFS (P = 0.023) but not for BCSS (P = 0.23). In the Cox hazards model, PA1 nuclear protein expression proved to be a significant prognostic univariate parameter for RFS (P = 0.03), but not for BCSS (P = 0.20). In addition, for those patients without lymphnode metastasis PA1 was found to be an independent prognostic factor for RFS (P = 0.025), which was verified by univariate and multivariate analyses. These investigations suggested PA1 expression could be a potential prognostic indicator for RFS in breast cancer.     

Chronic Academic Stress Increases a Group of microRNAs in Peripheral Blood

MicroRNAs (miRNAs) play key roles in regulation of cellular processes in response to changes in environment. In this study, we examined alterations in miRNA profiles in peripheral blood from 25 male medical students two months and two days before the National Examination for Medical Practitioners. Blood obtained one month after the examination were used as baseline controls. Levels of seven miRNAs (miR-16, -20b, -26b, -29a, -126, -144 and -144*) were significantly elevated during the pre-examination period in association with significant down-regulation of their target mRNAs (WNT4, CCM2, MAK, and FGFR1 mRNAs) two days before the examination. State anxiety assessed two months before the examination was positively and negatively correlated with miR-16 and its target WNT4 mRNA levels, respectively. Fold changes in miR-16 levels from two days before to one month after the examination were inversely correlated with those in WNT4 mRNA levels over the same time points. We also confirmed the interaction between miR-16 and WNT4 3′UTR in HEK293T cells overexpressing FLAG-tagged WNT4 3′UTR and miR-16. Thus, a distinct group of miRNAs in periheral blood may participate in the integrated response to chronic academic stress in healthy young men.     

Molecular and Clinical Studies in 138 Japanese Patients with Silver-Russell Syndrome

Background

Recent studies have revealed relative frequency and characteristic phenotype of two major causative factors for Silver-Russell syndrome (SRS), i.e. epimutation of the H19-differentially methylated region (DMR) and uniparental maternal disomy 7 (upd(7)mat), as well as multilocus methylation abnormalities and positive correlation between methylation index and body and placental sizes in H19-DMR epimutation. Furthermore, rare genomic alterations have been found in a few of patients with idiopathic SRS. Here, we performed molecular and clinical findings in 138 Japanese SRS patients, and examined these matters.

Methodology/Principal Findings

We identified H19-DMR epimutation in cases 1–43 (group 1), upd(7)mat in cases 44–52 (group 2), and neither H19-DMR epimutation nor upd(7)mat in cases 53–138 (group 3). Multilocus analysis revealed hyper- or hypomethylated DMRs in 2.4% of examined DMRs in group 1; in particular, an extremely hypomethylated ARHI-DMR was identified in case 13. Oligonucleotide array comparative genomic hybridization identified a ∼3.86 Mb deletion at chromosome 17q24 in case 73. Epigenotype-phenotype analysis revealed that group 1 had more reduced birth length and weight, more preserved birth occipitofrontal circumference (OFC), more frequent body asymmetry and brachydactyly, and less frequent speech delay than group 2. The degree of placental hypoplasia was similar between the two groups. In group 1, the methylation index for the H19-DMR was positively correlated with birth length and weight, present height and weight, and placental weight, but with neither birth nor present OFC.

Conclusions/Significance

The results are grossly consistent with the previously reported data, although the frequency of epimutations is lower in the Japanese SRS patients than in the Western European SRS patients. Furthermore, the results provide useful information regarding placental hypoplasia in SRS, clinical phenotypes of the hypomethylated ARHI-DMR, and underlying causative factors for idiopathic SRS.     

A Dominant-Negative FGF1 Mutant (the R50E Mutant) Suppresses Tumorigenesis and Angiogenesis

Fibroblast growth factor-1 (FGF1) and FGF2 play a critical role in angiogenesis, a formation of new blood vessels from existing blood vessels. Integrins are critically involved in FGF signaling through crosstalk. We previously reported that FGF1 directly binds to integrin αvβ3 and induces FGF receptor-1 (FGFR1)-FGF1-integrin αvβ3 ternary complex. We previously generated an integrin binding defective FGF1 mutant (Arg-50 to Glu, R50E). R50E is defective in inducing ternary complex formation, cell proliferation, and cell migration, and suppresses FGF signaling induced by WT FGF1 (a dominant-negative effect) in vitro. These findings suggest that FGFR and αvβ3 crosstalk through direct integrin binding to FGF, and that R50E acts as an antagonist to FGFR. We studied if R50E suppresses tumorigenesis and angiogenesis. Here we describe that R50E suppressed tumor growth in vivo while WT FGF1 enhanced it using cancer cells that stably express WT FGF1 or R50E. Since R50E did not affect proliferation of cancer cells in vitro, we hypothesized that R50E suppressed tumorigenesis indirectly through suppressing angiogenesis. We thus studied the effect of R50E on angiogenesis in several angiogenesis models. We found that excess R50E suppressed FGF1-induced migration and tube formation of endothelial cells, FGF1-induced angiogenesis in matrigel plug assays, and the outgrowth of cells in aorta ring assays. Excess R50E suppressed FGF1-induced angiogenesis in chick embryo chorioallantoic membrane (CAM) assays. Interestingly, excess R50E suppressed FGF2-induced angiogenesis in CAM assays as well, suggesting that R50E may uniquely suppress signaling from other members of the FGF family. Taken together, our results suggest that R50E suppresses angiogenesis induced by FGF1 or FGF2, and thereby indirectly suppresses tumorigenesis, in addition to its possible direct effect on tumor cell proliferation in vivo. We propose that R50E has potential as an anti-cancer and anti-angiogenesis therapeutic agent (“FGF1 decoy”).