An N-glycan structure correlates with pulmonary metastatic ability of cancer cells

N-Glycan structures on the surface of cancer cells have diverse structures and play significant roles in metastatic process. However, little is known about their roles in organ-selective metastasis. Our study revealed that an alpha1,6-fucosylated biantennary N-glycan structure designated A2G2F is characteristic of lungs, with far more abundant expression in normal human and murine lungs than in other organs. In this study, we further examined the role of A2G2F in pulmonary metastasis. We stained metastatic cancers by alpha1,6-fucose-specific Lens culinaris agglutinin lectin and revealed that pulmonary metastatic nodules more abundantly expressed alpha1,6-fucosylated N-glycans than hepatic metastatic nodules from common primary cancers. The most specific alpha1,6-fucosylated N-glycan structure in pulmonary metastatic cancer was identified to be A2G2F. Using a B16 melanoma cell metastasis model, we showed that A2G2F-rich B16 cells formed more pulmonary metastatic nodules than A2G2F-poor cells. Our results suggest that A2G2F plays a critical role in pulmonary metastasis.     

Femtoliter compartment in liposomes for in vitro selection of proteins

The aqueous compartment in liposomes provides a reaction resembling the cell and therefore is used as a microcompartment in which to study enzymatic reactions. However, regardless of their method of preparation, the heterogeneity in size of cell-size liposomes limits their potential uses. We established a strategy to estimate the internal aqueous volume of cell-size liposomes using a fluorescence-activated cell sorter (FACS). Reactions inside individual liposomes can be measured in a high-throughput format provided that the encapsulated proteins give rise to a fluorescent signal such as by exhibiting fluorescence themselves or by catalyzing production of a fluorescent compound. The strategy of volume estimation was applied to in vitro selection experiments. The green fluorescent protein (GFP) gene was encapsulated into liposomes together with an in vitro translation system. Here liposomes carrying a single copy of the gene were identified using the internal aqueous volume information of individual liposomes, and those exhibiting higher green fluorescence intensity were sorted by the FACS machine. This system was able to enrich those encoding GFP with higher fluorescence intensity over those with lower intensity. These results suggest the possibility of performing evolutionary experiments in an environment that mimics the cell.     

Lactococcin Q, a Novel Two-Peptide Bacteriocin Produced by Lactococcus lactis QU 4

A bacteriocin-producing strain, Lactococcus lactis QU 4, was isolated from corn. The bacteriocin, termed lactococcin Q, showed antibacterial activity only against L. lactis strains among a wide range of gram-positive indicator strains tested. Lactococcin Q was purified by acetone precipitation, cation exchange chromatography, and reverse-phase chromatography. Lactococcin Q consisted of two peptides, α and β, whose molecular masses were determined to be 4,260.43 Da and 4,018.36 Da, respectively. Amino acid and DNA sequencing analyses revealed that lactococcin Q was a novel two-peptide bacteriocin, homologous to lactococcin G. Comparative study using chemically synthesized lactococcin Q (Qα plus Qβ) and lactococcin G (Gα plus Gβ) clarified that hybrid combinations (Qα plus Gβ and Gα plus Qβ) as well as original combinations showed antibacterial activity, although each single peptide showed no significant activity. These four pairs of lactococcin peptides acted synergistically at a 1:1 molar ratio and exhibited identical antibacterial spectra but differed in MIC. The MIC of Qα plus Gβ was 32 times higher than that of Qα plus Qβ, suggesting that the difference in β peptides was important for the intensity of antibacterial activity.     

G protein-coupled receptor 30 is an estrogen receptor in the plasma membrane

Recently, GPR30 was reported to be a novel estrogen receptor; however, its intracellular localization has remained controversial. To investigate the intracellular localization of GPR30 in vivo, we produced four kinds of polyclonal antibodies for distinct epitopes on GPR30. Immunocytochemical observations using anti-GPR30 antibody and anti-FLAG antibody show that FLAG-GPR30 localizes to the plasma membrane 24 h after transfection. Treatment with estrogen (17beta-estradiol or E2) causes an elevation in the intracellular Ca2+ concentration ([Ca2+]i) within 10 s in HeLa cells expressing FLAG-GPR30. In addition, E2 induces the translocation of GPR30 from the plasma membrane to the cytoplasm by 1 h after stimulation. Immunohistochemical analysis shows that GPR30 exists on the cell surface of CA2 pyramidal neuronal cells. The images on transmission electron microscopy show that GPR30 is localized to a particular region associated with the plasma membranes of the pyramidal cells. These data indicate that GPR30, a transmembrane receptor for estrogen, is localized to the plasma membrane of CA2 pyramidal neuronal cells of the hippocampus in rat brain.     

The Caenorhabditis elegans homologue of deleted in azoospermia is involved in the sperm/oocyte switch

The Deleted in Azoospermia (DAZ) gene family encodes putative translational activators that are required for meiosis and other aspects of gametogenesis in animals. The single Caenorhabditis elegans homologue of DAZ, daz-1, is an essential factor for female meiosis. Here, we show that daz-1 is important for the switch from spermatogenesis to oogenesis (the sperm/oocyte switch), which is an essential step for the hermaphrodite germline to produce oocytes. RNA interference of the daz-1 orthologue in a related nematode, Caenorhabditis briggsae, resulted in a complete loss of the sperm/oocyte switch. The C. elegans hermaphrodite deficient in daz-1 also revealed a failure in the sperm/oocyte switch if the genetic background was conditional masculinization of germline. DAZ-1 could bind specifically to mRNAs encoding the FBF proteins, which are translational regulators for the sperm/oocyte switch and germ stem cell proliferation. Expression of the FBF proteins seemed to be lowered in the daz-1 mutant at the stage for the sperm/oocyte switch. Conversely, a mutation in gld-3, a gene that functionally counteracts FBF, could partially restore oogenesis in the daz-1 mutant. Together, we propose that daz-1 plays a role upstream of the pathway for germ cell sex determination.     

Reactive oxygen species mediate shear stress-induced fluid-phase endocytosis in vascular endothelial cells

To elucidate the role of shear stress in fluid-phase endocytosis of vascular endothelial cells (EC), we used a rotating-disk shearing apparatus to investigate the effects of shear stress on the uptake of lucifer yellow (LY) by cultured bovine aortic endothelial cells (BAEC). Exposure of EC to shear stress (area-mean value of 10 dynes/cm2) caused an increase in LY uptake that was abrogated by the antioxidant, N-acetyl-L-cysteine (NAC), the NADPH oxidase inhibitor, acetovanillone, and two inhibitors of protein kinase C (PKC), calphostin C and GF109203X. These results suggest that fluid-phase endocytosis is regulated by both reactive oxygen species (ROS) and PKC. Shear stress increased both ROS production and PKC activity in EC, and the increase in ROS was unaffected by calphostin C or GF109203X, whereas the activation of PKC was reduced by NAC and acetovanillone. We conclude that shear stress-induced increase in fluid-phase endocytosis is mediated via ROS generation followed by PKC activation in EC.     

Interaction of KAI1 on tumor cells with DARC on vascular endothelium leads to metastasis suppression

CD82, also known as KAI1, was recently identified as a prostate cancer metastasis suppressor gene on human chromosome 11p1.2 (ref. 1). The product of CD82 is KAI1, a 40- to 75-kDa tetraspanin cell-surface protein also known as the leukocyte cell-surface marker CD82 (refs. 1,2). Downregulation of KAI1 has been found to be clinically associated with metastatic progression in a variety of cancers, whereas overexpression of CD82 specifically suppresses tumor metastasis in various animal models. To define the mechanism of action of KAI1, we used a yeast two-hybrid screen and identified an endothelial cell-surface protein, DARC (also known as gp-Fy), as an interacting partner of KAI1. Our results indicate that the cancer cells expressing KAI1 attach to vascular endothelial cells through direct interaction between KAI1 and DARC, and that this interaction leads to inhibition of tumor cell proliferation and induction of senescence by modulating the expression of TBX2 and p21. Furthermore, the metastasis-suppression activity of KAI1 was significantly compromised in DARC knockout mice, whereas KAI1 completely abrogated pulmonary metastasis in wild-type and heterozygous littermates. These results provide direct evidence that DARC is essential for the function of CD82 as a suppressor of metastasis.     

Chronic intracerebroventricular administration of relaxin-3 increases body weight in rats

Bolus-administered intracerebroventricular (ICV) relaxin-3 has been reported to increase feeding. In this study, to examine the role of relaxin-3 signaling in energy homeostasis, we studied the effects of chronically administered ICV relaxin-3 on body weight gain and locomotor activity in rats. Two groups of animals received vehicle or relaxin-3 at 600 pmol/head/day, delivered with Alzet osmotic minipumps. In animals receiving relaxin-3, food consumption and weight gain were statistically significantly higher than those in the vehicle group during the 14-day infusion. During the light phase on days 2 and 7 and the dark phase on days 3 and 8, there was no difference in locomotor activity between the two groups. Plasma concentrations of leptin and insulin in rats chronically injected with relaxin-3 were significantly higher than in the vehicle-injected controls. These results indicate that relaxin-3 up-regulates food intake, leading to an increase of body weight and that relaxin-3 antagonists might be candidate antiobesity agents.     

Cdk5 phosphorylates and stabilizes p27kip1 contributing to actin organization and cortical neuronal migration

p27(kip1), a cyclin-dependent kinase (CDK) inhibitor (CKI), generally suppresses CDK activity in proliferating cells. Although another role of p27 in cell migration has been recently suggested in vitro, the physiological importance of p27 in cell migration remains elusive, as p27-deficient mice have not shown any obvious migration-defect-related phenotypes. Here, we show that Cdk5, an unconventional neuronal CDK, phosphorylates and stabilizes p27 as an upstream regulator, maintaining the amount of p27 in post-mitotic neurons. In vivo RNA interference (RNAi) experiments showed that reduced amounts of p27 caused inhibition of cortical neuronal migration and decreased the amount of F-actin in the processes of migrating neurons. The Cdk5-p27 pathway activates an actin-binding protein, cofilin, which is also shown to be involved in cortical neuronal migration in vivo. Our findings shed light on a previously unknown new relationship between CDK and CKI in G0-arrested cells that regulates cytoskeletal reorganization and neuronal migration during corticogenesis.     

Identification of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible genes in human amniotic epithelial cells

Background  

Exposure to dioxins results in a broad range of pathophysiological disorders in human fetuses. In order to evaluate the effects of dioxins on the feto-placental tissues, we analyzed the gene expression in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated primary cultures of human amniotic epithelial cells.