Regulation of N-cadherin-based cell–cell interaction by JSAP1

金沢大学がん研究所がん分子細胞制御金沢大学大学院医学系研究科We previously reported that the level of c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein for JNK signaling, increases dramatically during nerve growth factor (NGF)-induced differentiation of PC12h cells. In the present study, we investigated the function of JSAP1 during PC12h cell differentiation by knocking down the level of JSAP1. The depletion of JSAP1 caused NGF-treated PC12h cells to form aggregates and impaired their differentiation. The aggregation was not observed in JSAP1-depleted cells that were untreated or treated with epidermal growth factor. Immunocytochemical studies indicated that N-cadherin, but not E-cadherin, was localized to sites of cell–cell contact in the aggregated cells. Furthermore, an inhibitory anti-N-cadherin antibody completely blocked the aggregation. Taken together, these results suggest that JSAP1 regulates cell–cell interactions in PC12h cells specifically in the NGF-induced signaling pathway, and does so by modulating N-cadherin

The Scaffold Protein c-Jun NH2-Terminal Kinase-associated Leucine Zipper Protein Regulates Cell Migration through Interaction with the G Protein G13

金沢大学がん研究所がん分子細胞制御Scaffold proteins for MAP kinase (MAPK) signalling modules play an important role in the specific and efficient signal transduction of the relevant MAPK cascades. Here, we investigated the function of the scaffolding protein c-Jun NH2-terminal kinase (JNK)-associated leucine zipper protein (JLP) by depleting it in cultured cells using a short hairpin RNA (shRNA) against human JLP. HeLa and DLD-1 cells stably expressing the shRNA showed a defect in cell migration. The re-expression of full-length shRNA-resistant mouse JLP rescued the impaired cell migration of the JLP-depleted HeLa cells; whereas, a C-terminal deletion mutant of mouse JLP, which failed to bind the G protein Gα13, showed little or no effect on the cell migration defect. Furthermore, although a constitutively active Gα13 enhanced the migration of control HeLa cells, the Gα13-induced cell migration was significantly suppressed in the JLP-depleted HeLa cells. Taken together, these results suggest that JLP regulates cell migration through an interaction with Gα13. © The Authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Assessment of genetic diversity in sea buckthorn (Hippophae rhamnoides L.) accessions from Mongolia based on RAPD markers

Hippophae rhamnoides L., which belongs to the Elaegnaceae family, is one of the medically and environmentally valuable berry crops with its high nutritious and bioactive compounds. Despite its high demand in the food, medicinal and agricultural industries, this species has been less studied molecularly. In view of this, an effort has been made in the present study to characterize 24 accessions of H. rhamnoides collected from different geographical regions of Mongoliaa through random amplified polymorphic DNA (RAPD) markers. A total of 10 RAPD primers were used in the present study for their ability to produce clear, scorable amplicons. The RAPD analysis totally generated 87 bands, of which 84 (96.34%) were polymorphic, pointing to a high degree of genetic variation. The similarity coefficient ranged from 0.4-1 with the mean of 0.78. The UPGMA dendrogram was generated using these data grouped accessions into two main clusters. Cluster analysis reflected a relatively close relationship between accessions grown at the same or neighbouring areas. Thus, our data could be informative for further selection and management of germplasm collections and crossing strategies for sea buckthorn

Development of serological assay for detection of antibodies in the N protein of SARS-CoV-2 in human sera in Mongolia

The capability to detect Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and identify immune responses among the population is crucial for managing the outbreak of the COVID-19 pandemic. Although PCR-based nucleic acid detection techniques are utilized to detect viral infection in people, alternative tests capable of distinguishing between exposure and infection are urgently needed beyond this restricted window of detectable viral replication. Antibodies are produced in human sera within a few days after viral infection, providing longer period for performing tests to acquire reliable database. Herewith, we provide the results of our in-house developed ELISA (Enzyme-Linked Immunosorbent Assay) that displays all of the properties necessary for high-throughput of human sera sample analysis. This test does not involve the handling of live viruses, although it detects a variety of antibody types in serum and plasma of human after exposure to the virus. For in-house development of the kit, the nucleocapsid (N) gene of SARS-CoV-2 virus was cloned in the prokaryotic expression vector pGEX-6P-1, and purified N protein was used to detect IgG antibodies in human sera samples. In total 76 human serum samples that were collected before novel coronavirus registry in Mongolia in March 2020, as well as 200 serum samples from patients who had been infected by SARS-CoV-2 virus, were used. Among 200 serum samples, 188 were positive and 12 were false negative, while in non-infected cases 69 were negative and 7 were false positive, suggesting 94 per cent sensitivity and 90.7 per cent specificity of the kit, with p-values of 0.02

AKT and 14-3-3 regulate Notch4 nuclear localization

Members of the Notch family of transmembrane receptors, Notch1-4 in mammals, are involved in the regulation of cell fate decisions and cell proliferation in various organisms. The Notch4 isoform, which is specific to mammals, was originally identified as a viral oncogene in mice, Int3, able to initiate mammary tumors. In humans, Notch4 expression appears to be associated with breast cancer stem cells and endocrine resistance. Following ligand binding, the Notch4 receptor undergoes cleavage at the membrane and the Notch4-intracellular domain (ICD), translocates to the nucleus and regulates gene transcription. Little is known on the mechanisms regulating Notch4-ICD and its nuclear localization. Here, we describe the identification of four distinct AKT phosphorylation sites in human Notch4-ICD and demonstrate that AKT binds Notch4-ICD and phosphorylates all four sites in vitro and in vivo. The phosphorylation in cells is regulated by growth factors and is sensitive to phosphatidyl inositol-3 kinase (PI3K) inhibitors. This phosphorylation generates binding sites to the 14-3-3 regulatory proteins, which are involved in the regulation of nucleocytoplasmic shuttling of target proteins, restricting phosphorylated Notch4-ICD to the cytoplasm. Our findings provide a novel mechanism for Notch4-ICD regulation, suggesting a negative regulatory role for the PI3K-AKT pathway in Notch4 nuclear signaling

Ablation of the scaffold protein JLP causes reduced fertility in male mice

金沢大学がん研究所がん分子細胞制御The specific and efficient activation of mitogen-activated protein kinase (MAPK) signaling modules is mediated, at least in part, by scaffold proteins. c-Jun NH2-terminal kinase (JNK)-associated leucine zipper protein (JLP) was identified as a scaffold protein for JNK and p38 MAPK signaling modules. JLP is expressed nearly ubiquitously and is involved in intracellular signaling pathways, such as the Gα13 and Cdo-mediated pathway, in vitro. To date, however, JLP expression has not been analyzed in detail, nor are its physiological functions well understood. Here we investigated the expression of JLP in the mouse testis during development. Of the tissues examined, JLP was strongest in the testis, with the most intense staining in the elongated spermatids. Since the anti-JLP antibody used in this study can recognize both JLP and sperm-associated antigen 9 (SPAG9), a splice variant of JLP that has been studied extensively in primates, we also examined its expression in macaque testis samples. Our results indicated that in mouse and primate testis, the isoform expressed at the highest level was JLP, not SPAG9. We also investigated the function of JLP by disrupting the Jlp gene in mice, and found that the male homozygotes were subfertile. Taken together, these observations may suggest that JLP plays an important role in testis during development, especially in the production of functionally normal spermatozoa. © 2008 Springer Science+Business Media B.V

Antibacterial activity of Arthrobacter strains isolated from Great Gobi A Strictly Protected Area, Mongolia

Desert soil hosts many microorganisms, whose activities are essential from an ecological viewpoint. Moreover, they are of great anthropic interest. The knowledge of extreme environments microbiomes may be beneficial for agriculture, technology, and human health. In this study, 11 Arthrobacter strains from topsoil samples collected from the Great Gobi A Strictly Protected Area in the Gobi Desert, were characterized by a combination of different techniques. The phylogenetic analysis, performed using their 16S rDNA sequences and the most similar Arthrobacter sequences found in databases, revealed that most of them were close to A. crystallopoietes, while others joined a sister group to the clade formed by A. humicola, A. pascens, and A. oryzae. The resistance of each strain to different antibiotics, heavy -metals, and NaCl was also tested as well as the inhibitory potential against human pathogens (i.e., Burkholderia ssp., Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus ssp.) via cross -streaking, to check the production of metabolites with antimicrobial activity. Data obtained revealed that all strains were resistant to heavy metals and were able to strongly interfere with the growth of many of the human pathogens tested. The volatile organic compounds (VOCs) profile of the 11 Arthrobacter strains was also analyzed. A total of 16 different metabolites were found, some of which were already known for having an inhibitory action against different Grampositive and Gram-negative bacteria. Isolate MS -3A13, producing the highest quantity of VOCs, is the most efficient against Burkholderia cepacia complex (Bcc), K. pneumoniae, and coagulase-negative Staphylococci (CoNS) strains. This work highlights the importance of understanding microbial populations' phenotypical characteristics and dynamics in extreme environments to uncover the antimicrobial potential of new species and strains