Practical methodology for gametophyte proliferation and sporophyte production in green penny fern (Lemmaphyllum microphyllum C. Presl) using mechanical fragmentation

Propagation of gametophytes and sporophytes using mechanical fragmentation has been considered a suitable method for mass production of ferns. This study aimed to develop a practical propagation method for Lemmaphyllum microphyllum C. Presl, which is a fern of significant ornamental and medicinal value. Gametophytes were obtained through in vitro spore germination and used for propagation experiments. The gametophyte was mechanically fragmented using a scalpel into small fragments, which were then used to investigate gametophyte proliferation. In addition, the gametophyte was fragmented using a blender and then used to study sporophyte formation. Optimal proliferation conditions of the gametophyte were determined using Murashige and Skoog (MS) basal medium (double-, full-, half-, quarter-strength), Knop medium, and medium components (sucrose, nitrogen sources, activated charcoal), at various concentrations. The fresh weight of the gametophyte was 14-fold higher than that of gametophytes (300 mg) used as culture material, when cultured on double-strength MS. Moreover, 1 g of the gametophyte fragmented in 25 mL of distilled water formed more than 430 sporophytes in a soil mixture in an area of 7.5 cm². The sporophytes were successfully cultivated in the greenhouse after acclimation. A large-scale production method for L. microphyllum that can be easily implemented in a fern production farm is outlined.

     

Cytoskeleton‐associated proteins are enriched in human embryonic‐stem cell‐derived neuroectodermal spheres

The ability to generate neural lineages from human embryonic stem cells (hESCs) in a controlled manner would further investigation of human neurogenesis and development of potential cell therapeutic applications to treat neurological diseases; however, generating such neural stem cells (NSCs) remains a challenge. In an attempt to characterize the cellular mechanisms involved in hESC differentiation into neuroprogenitor cells, we performed 2‐DE using protein extracts from hESC‐derived embryoid bodies (EBs) and neuroectodermal spheres (NESs) bearing neuroprogenitors. Of 47 differentially expressed protein spots, 28 nonredundant spots were shown to be upregulated in the NESs; these protein spots included neurogenesis‐related proteins (TAF1, SEPT2, NPH3, and CRABP), as expected. Interestingly, 6 of these 28 protein spots were cytoskeleton‐associated proteins (CSAP) such as Fascin‐1, Cofilin‐1, and Stathmin‐1. Western‐blot analyses confirmed the increased levels of these proteins in the NESs. Furthermore, immunostaining analysis showed that both Fascin‐1 and Stathmin‐1 were preferentially expressed in the inner rims of neural rosettes, which are characteristic features of neuroprogenitors in culture. We also confirmed prominent expression of Fascin‐1 in (sub‐)ventricular zone in E15.5 mouse fetal brain. Our results suggest that, in addition to the induction of those genes involved in neural development, hESC differentiation into the NES is associated with a marked reorganization of the cellular cytoskeleton.     

Aβ1–42 reduces P-glycoprotein in the blood–brain barrier through RAGE–NF-κB signaling

The reduced clearance of amyloid-β peptide (Aβ) from the brain partly accounts for the neurotoxic accumulation of Aβ in Alzheimer''s disease (AD). Recently, it has been suggested that P-glycoprotein (P-gp), which is an efflux transporter expressed on the luminal membrane of the brain capillary endothelium, is capable of transporting Aβ out of the brain. Although evidence has shown that restoring P-gp reduces brain Aβ in a mouse model of AD, the molecular mechanisms underlying the decrease in P-gp expression in AD is largely unknown. We found that Aβ_1–42 reduced P-gp expression in the murine brain endothelial cell line bEnd.3, which was consistent with our in vivo data that P-gp expression was significantly reduced, especially near amyloid plaques in the brains of five familial AD mutations (5XFAD) mice that are used as an animal model for AD. A neutralizing antibody against the receptor for advanced glycation end products (RAGE) and an inhibitor of nuclear factor-kappa B (NF-κB) signaling prevented the decrease in Aβ_1–42-induced P-gp expression, suggesting that Aβ reduced P-gp expression through NF-κB signaling by interacting with RAGE. In addition, we observed that the P-gp reduction by Aβ was rescued in bEnd.3 cells receiving inductive signals or factors from astrocytes making contacts with endothelial cells (ECs). These results support that alterations of astrocyte–EC contacts were closely associated with P-gp expression. This suggestion was further supported by the observation of a loss of astrocyte polarity in the brains of 5XFAD mice. Taken together, we found that P-gp downregulation by Aβ was mediated through RAGE–NF-κB signaling pathway in ECs and that the contact between astrocytes and ECs was an important factor in the regulation of P-gp expression.Alzheimer''s disease (AD) is a neurodegenerative disorder that is characterized by a progressive loss of cognitive function leading to dementia. The major pathological hallmark of AD is the deposition of neurotoxic amyloid-β peptide (Aβ) within the brain.¹ The amyloid hypothesis proposes that the accumulation of Aβ is caused by an imbalance between Aβ production and clearance.² Although genetic alterations increase the production of Aβ in rare familial AD, reduced Aβ clearance from the brain likely accounts for sporadic AD, which is much more common.³ The mechanisms that are involved in clearing Aβ from the brain include enzymatic degradation, perivascular drainage, and the most significant, active transport across the blood–brain barrier (BBB).⁴The BBB regulates molecular exchanges at the interface between the blood and the brain.⁵ It plays a critical role in maintaining the brain microenvironment.⁶ The BBB, which is formed by cerebral endothelial cells (ECs) and which, interacts with astrocytes, neurons, pericytes, and the extracellular matrix, is organized into a neurovascular unit.^{7, 8} Although the relationship between BBB breakdown and AD pathology is unclear,⁹ it has been proposed that the BBB loses its Aβ clearing capability, thus increasing amyloid deposition in the outer capillary membrane and resulting in the distortion of the neurovascular unit with neuronal loss.¹⁰Recently, it has been suggested that P-glycoprotein (P-gp), which is an ATP-driven efflux transporter that is highly expressed in the luminal membrane of the brain capillary endothelium, is also involved in the clearance of Aβ from the brain.¹¹ P-gp, which is able to transport various kinds of substrates, has been shown to play an important role in clearing toxic substances in the brain and protecting it from harmful molecules in the circulation.¹² Along with other BBB properties, P-gp expression is induced when ECs are in contact with astrocytes in vitro and in vivo.^{13, 14} ECs respond to inductive signals or factors from astrocytes that encircle the capillary endothelium.¹³Several lines of evidence have shown that P-gp plays an important role in Aβ clearance. It has been shown in vitro that P-gp mediates the transport of Aβ and that blocking P-gp function reduces the clearance of Aβ.^{15, 16} In addition, cerebral Aβ deposition in elderly non-demented individuals has been demonstrated to be inversely correlated with brain capillary P-gp expression.¹⁷ Furthermore, in P-gp knockout mice, Aβ deposition is increased by the reduced efflux of Aβ,¹⁸ while it has been shown that restoring P-gp at the BBB reduces brain Aβ in a mouse model of AD.¹⁹ However, the molecular mechanisms underlying the decrease in P-gp expression that is observed in AD have not been identified. We found that Aβ decreased P-gp expression by increasing nuclear factor-kappa B (NF-κB) through an interaction with the receptor for advanced glycation end products (RAGE). Moreover, we observed that the P-gp reduction by Aβ was rescued by inductive signals or factors from astrocytes that made contact with ECs in bEnd.3 cells. These results suggested that alterations in astrocyte–EC contact in AD likely decrease P-gp expression by Aβ. Together, we identified a mechanism by which the Aβ–RAGE interaction mediated the downregulation of P-gp in the BBB by increasing NF-κB signaling in AD and that astrocyte–EC contact played a critical role in maintaining P-gp expression.     

Mycobacterium paraterrae sp. nov. recovered from a clinical specimen: novel chromogenic slow growing mycobacteria related to Mycobacterium terrae complex

A previously unidentified, slowly growing scotochromogenic Mycobacterium was isolated from a Korean patient with symptomatic pulmonary infection. Phenotypically, this strain was generally similar to Mycobacterium terrae complex strains, however it uniquely produced orange pigmentation. Unique mycolic acid profiles and phylogenetic analyses based on three alternative chronometer molecules, 16S rRNA gene, hsp65 and rpoB , confirmed the taxonomic status of this strain as a novel species. These results support that this strain represents a novel Mycobacterium species. The name Mycobacterium paraterrae sp. nov. is proposed. The type strain is 05-2522 (= DSM 45127 = KCTC 19556).     

Prolonged gene expression in primary porcine pancreatic cells using an Epstein-Barr virus-based episomal vector

Epstein-Barr virus (EBV)-based plasmids containing the origin of replication (oriP) and EBV nuclear antigen 1 (EBNA-1) are well known for the stable episomal maintenance in human cells. In order to clarify whether an EBV-based plasmid can be maintained stably in the porcine pancreatic cells which are the primary candidate sources of islet xenotransplantation, we constructed pEBVGFP encoding the green fluorescent protein (GFP). Monolayer culture of the porcine neonatal pancreatic cells was lipofected with pEBVGFP or pGFP which was derived from pEBVGFP by deleting out oriP and EBNA-1. pEBVGFP significantly prolonged GFP expression not only in human cell lines but also in the primary porcine pancreatic cells compared with pGFP. Interestingly, the duct cells that are believed as the pancreatic precursor cells were preferentially transfected and conveniently enriched among the mixed primary cell populations using a hygromycin B selection. To our knowledge, this is the first report suggesting the potential application of an EBV-based plasmid for the extended gene expression in the primary porcine pancreatic duct cells.     

Sphingomonas kyeonggiense sp. nov., isolated from soil of a ginseng field

A Gram-staining negative bacterium, THG-DT81^T, which was isolated from soil of a ginseng field, was investigated using a polyphasic taxonomic approach. Cells were oxidase- and catalase-positive, aerobic, rod-shaped and motile with one polar flagellum. Strain THG-DT81^T grew optimally at pH 7.0 and in the absence of NaCl on trypticase soy agar. Its optimum growth temperature was 25–28 °C. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain THG-DT81^T belongs to the family Sphingomonadaceae and was related to Sphingomonas pituitosa EDIV^T (98.0 % similarity), S. leidyi ATCC 15260^T (97.8 %), S. trueperi LMG 2142^T (97.1 %), S. azotifigens NBRC 15497^T (97.1 %), S. koreensis JSS26 ^T (97.1 %) and S. dokdonensis DS-4^T (97.0 %). Strain THG-DT81^T contained Q-10 as the predominant ubiquinone and C_18:1 ω7c and C_16:0 as the major fatty acids. The G+C content of the genomic DNA was determined to be 66.8 mol %. The major component in the polyamine pattern was identified as sym-homospermidine. The major polar lipids detected in strain THG-DT81^T were sphingoglycolipid, phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylcholine. The DNA–DNA relatedness values of the strain THG-DT81^T and its closest phylogenetically neighbors were below 21 %. The phenotypic characteristics and genotypic data demonstrated the affiliation of strain THG-DT81^T to the genus Sphingomonas. On the basis of the polyphasic taxonomic data presented, strain THG-DT81^T is described as a novel species of genus Sphingomonas, for which the name Sphingomonas kyeonggiense sp. nov. is proposed. The type strain is THG-DT81^T (= KACC 17173^T = JCM 18825^T).     

Role of receptor-attached phosphates in binding of visual and non-visual arrestins to G protein-coupled receptors

Arrestins are a small family of proteins that regulate G protein-coupled receptors (GPCRs). Arrestins specifically bind to phosphorylated active receptors, terminating G protein coupling, targeting receptors to endocytic vesicles, and initiating G protein-independent signaling. The interaction of rhodopsin-attached phosphates with Lys-14 and Lys-15 in β-strand I was shown to disrupt the interaction of α-helix I, β-strand I, and the C-tail of visual arrestin-1, facilitating its transition into an active receptor-binding state. Here we tested the role of conserved lysines in homologous positions of non-visual arrestins by generating K2A mutants in which both lysines were replaced with alanines. K2A mutations in arrestin-1, -2, and -3 significantly reduced their binding to active phosphorhodopsin in vitro. The interaction of arrestins with several GPCRs in intact cells was monitored by a bioluminescence resonance energy transfer (BRET)-based assay. BRET data confirmed the role of Lys-14 and Lys-15 in arrestin-1 binding to non-cognate receptors. However, this was not the case for non-visual arrestins in which the K2A mutations had little effect on net BRET(max) values for the M2 muscarinic acetylcholine (M2R), β(2)-adrenergic (β(2)AR), or D2 dopamine receptors. Moreover, a phosphorylation-deficient mutant of M2R interacted with wild type non-visual arrestins normally, whereas phosphorylation-deficient β(2)AR mutants bound arrestins at 20-50% of the level of wild type β(2)AR. Thus, the contribution of receptor-attached phosphates to arrestin binding varies depending on the receptor-arrestin pair. Although arrestin-1 always depends on receptor phosphorylation, its role in the recruitment of arrestin-2 and -3 is much greater in the case of β(2)AR than M2R and D2 dopamine receptor.