Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified,long shelf-life polyethylenimine

Introduction of genetic material into cells is an essential prerequisite for current research in molecular cell biology. Although transfection with commercially available reagents results in excellent gene expression, their high costs are obstacles to experimentation with a large number or large scales of transfection. The cationic polymer linear-polyethylenimine (MW 25,000) (PEI), one of the most cost-effective vehicles, facilitates DNA compaction by polyplex formation, which leads to efficient delivery of DNA into cells by endocytosis. However, the use of PEI is still limited because of substantial cytotoxicity and intolerable deterioration in transfection efficiency by its low stability. Here, we show that acidification of PEI is important for its transfection activity. Dissolving PEI powder in 0.2N HCl confers a long shelf-life for PEI storage at 4 and −80 °C, and the polyplex formation of plasmid DNA with PEI is optimized in lactate-buffered saline at pH 4.0. Furthermore, changing the culture medium at 8–12 h posttransfection can minimize the cytotoxicity of PEI without sacrificing the high transfection efficiency comparable to that of commercial reagents. The cost per test using acidified PEI is drastically reduced to approximately 1:10,000, compared with commercial reagents. Thus, we conclude that acidification of PEI satisfactorily accomplishes cost-effective, high-efficiency transfection.     

Isolation and mapping of a new set of 129 RFLP markers in Arabidopsis thaliana using recombinant inbred lines

A new collection of 129 Arabidopsis thaliana RFLP markers has been established based upon DNA fragments cloned in the pUC119 plasmid vector and insert end sequences of P1 clones. Dominant/null alleles affecting low-copy number sequences account for nine of the mapped polymorphisms, suggesting that deletions are not rare in A. thaliana . Recombinant inbred (RI) lines were used for mapping these marker loci. RI line-based mapping allows integration of this set of markers with markers previously reported as well as with any markers mapped in the future using this replenishable mapping resource. These markers are useful for map-based gene isolation and genome physical mapping in A. thaliana as well as studies of chromosome colinearity (synteny) with related species.     

Visualization of Sialidase Activity in Mammalian Tissues and Cancer Detection with a Novel Fluorescent Sialidase Substrate

Sialidase removes sialic acid from sialoglycoconjugates and plays crucial roles in many physiological and pathological processes. Various human cancers express an abnormally high level of the plasma membrane-associated sialidase isoform.Visualization of sialidase activity in living mammalian tissues would be useful not only for understanding sialidase functions but also for cancer diagnosis. However, since enzyme activity of mammalian sialidase is remarkably weak compared with that of bacterial and viral sialidases, it has been difficult to detect sialidase activity in mammalian tissues. We synthesized a novel benzothiazolylphenol-based sialic acid derivative (BTP-Neu5Ac) as a fluorescent sialidase substrate. BTP-Neu5Ac can visualize sialidase activities sensitively and selectively in acute rat brain slices. Cancer cells implanted orthotopically in mouse colons and human colon cancers (stages T3-T4) were also clearly detected with BTP-Neu5Ac. The results suggest that BTP-Neu5Ac is useful for histochemical imaging of sialidase activities.     

Requirement of the SH4 and tyrosine-kinase domains but not the kinase activity of Lyn for its biosynthetic targeting to caveolin-positive Golgi membranes

Background

The Src-family non-receptor-type tyrosine kinase Lyn, which is often associated with chemotherapeutic resistance in cancer, localizes not only to the plasma membrane but also Golgi membranes. Recently, we showed that Lyn, which is synthesized in the cytosol, is transported from the Golgi to the plasma membrane along the secretory pathway. However, it is still unclear how Golgi targeting of newly synthesized Lyn is regulated.

Methods

Subcellular localization of Lyn and its mutants was determined by confocal microscopy.

Results

We show that the kinase domain, but not the SH3 and SH2 domains, of Lyn is required for the targeting of Lyn to the Golgi, whereas the N-terminal lipids of the Lyn SH4 domain are not sufficient for its Golgi targeting. Although intact Lyn, which colocalizes with caveolin-positive Golgi membranes, can traffic toward the plasma membrane, kinase domain-deleted Lyn is immobilized on caveolin-negative Golgi membranes.

General significance

Besides the SH4 domain, the Lyn kinase domain is important for targeting of newly synthesized Lyn to the Golgi, especially caveolin-positive transport membranes. Our results provide a novel role of the Lyn catalytic domain in the Golgi targeting of newly synthesized Lyn in a manner independent of its kinase activity.     

Junctional adhesion molecule-C promotes metastatic potential of HT1080 human fibrosarcoma

The junctional adhesion molecule (JAM) family is a key molecule in a process called transendothelial migration or diapedesis. Here, we report implications of JAM-C in cancer metastasis. We first determined the mRNA expression of JAMs in 19 kinds of cancer cell lines. JAM-C was expressed in most of tumors having potent metastatic properties. Especially in murine K-1735 melanoma cell lines, the highly metastatic sublines (M2 and X21) strongly expressed JAM-C when compared with the poorly metastatic ones (C-10 and C23). Next, we investigated the role of JAM-C in cancer metastasis by using human JAM-C (hJAM-C) gene-transfected HT1080 fibrosarcoma cells. In comparison with mock-transfected HT1080 cells, these cells showed a significant increase in the adhesion to various extracellular substrates and the invasion across a Matrigel-coated membrane. The knockdown of hJAM-C using small interfering RNA resulted in the suppression of both the adhesion and the invasion of HT1080 cells, suggesting that endogenous hJAM-C might be involved in tumor metastasis. Finally, we studied the role of hJAM-C in an in vivo experimental metastatic model. The results showed that the overexpression of hJAM-C in HT1080 cells significantly decreased the life spans of the tumorbearing mice. In contrast, the knockdown of hJAM-C in HT1080 cells suppressed the weight gain of the lungs with metastatic colonies. We conclude that the expression of JAM-C promotes metastasis by enhancing both the adhesion of cancer cells to extracellular matrices and the subsequent invasion.     

Infectivity of Chlorella species for the ciliate Paramecium bursaria is not based on sugar residues of their cell wall components, but on their ability to localize beneath the host cell membrane after escaping from the host digestive vacuole in the early infection process

Summary. Paramecium bursaria cells harbor several hundred symbiotic algae in their cytoplasm. Algae-free cells can be reinfected with algae isolated from algae-bearing cells or cultivated Chlorella species through the digestive vacuoles. To determine the relationship between the infectivity of various Chlorella species and the nature of their cell wall components, algae-free P. bursaria cells were mixed with 15 strains of cultivated Chlorella species and observed for the establishment of endosymbiosis at 1 h and 3 weeks after mixing. Only 2 free-living algal strains, C. sorokiniana C-212 and C. kessleri C-531, were maintained in the host cells, whereas free-living C. sorokiniana C-43, C. kessleri C-208, C. vulgaris C-27, C. ellipsoidea C-87 and C-542, C. saccharophila C-183 and C-169, C. fusca var. vacuolata C-104 and C-28, C. zofingiensis C-111, and C. protothecoides C-150 and C-206 and the cultivated symbiotic Chlorella sp. strain C-201 derived from Spongilla fluviatilis could not be maintained. These infection-incapable strains could escape from the host digestive vacuole but failed to localize beneath the host cell membrane and were eventually digested. Labeling of their cell walls with Alexa Fluor 488-conjugated wheat germ agglutinin, GS-II, or concanavalin A, with or without pretreatment with 0.4 N NaOH, showed no relationship between their infectivity and the stainability with these lectins. Our results indicate that the infectivity of Chlorella species for P. bursaria is not based on the sugar residues on their cell wall and on the alkali-insoluble part of the cell wall components, but on their ability to localize just beneath the host cell membrane after escaping from the host digestive vacuole. Correspondence and reprints: Environmental Science and Engineering, Graduate School of Science and Engineering, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan.     

Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts

Estrogen prevents osteoporotic bone loss by attenuating bone resorption; however, the molecular basis for this is unknown. Here, we report a critical role for the osteoclastic estrogen receptor alpha (ERalpha) in mediating estrogen-dependent bone maintenance in female mice. We selectively ablated ERalpha in differentiated osteoclasts (ERalpha(DeltaOc/DeltaOc)) and found that ERalpha(DeltaOc/DeltaOc) females, but not males, exhibited trabecular bone loss, similar to the osteoporotic bone phenotype in postmenopausal women. Further, we show that estrogen induced apoptosis and upregulation of Fas ligand (FasL) expression in osteoclasts of the trabecular bones of WT but not ERalpha(DeltaOc/DeltaOc) mice. The expression of ERalpha was also required for the induction of apoptosis by tamoxifen and estrogen in cultured osteoclasts. Our results support a model in which estrogen regulates the life span of mature osteoclasts via the induction of the Fas/FasL system, thereby providing an explanation for the osteoprotective function of estrogen as well as SERMs.     

Distribution,body size and biology of the megamouth shark Megachasma pelagios

We compiled historical reports of megamouth sharks Megachasma pelagios (mostly fishery by-catch and strandings) from 1976 to 2018 (n = 117) and found that they are distributed globally (highest latitude, 36°) with three hotspots: Japan, Taiwan and the Philippines. Despite possible biases due to variability in fishing effort, more individuals were reported at higher latitudes in the summer, suggesting seasonal, latitudinal migrations. Sex ratios were female-biased in Japan, but more even in Taiwan and the Philippines, suggesting some sexual segregation. Females (total length, L_T = 3.41–7.10 m) were larger than males (L_T = 1.77–5.39 m) and matured at a larger L_T (5.17 m) than males (4.26 m). Also, we reviewed the systematics, feeding ecology and swimming behaviour of Megachasma pelagios based on the literature. Our review shows that, compared with their morphology, anatomy and genetics, behavioural ecology of this species remains largely unknown and electronic tagging studies are warranted.     

Orexin/Hypocretin Activates mTOR Complex 1 (mTORC1) via an Erk/Akt-independent and Calcium-stimulated Lysosome v-ATPase Pathway

The lack of the neuropeptide orexin, also known as hypocretin, results in narcolepsy, a chronic sleep disorder characterized by frequent sleep/cataplexy attacks and rapid eye movement sleep abnormalities. However, the downstream pathways of orexin signaling are not clearly understood. Here, we show that orexin activates the mTOR pathway, a central regulator of cell growth and metabolism, in the mouse brain and multiple recombinant cell lines that express the G protein-coupled receptors (GPCRs), orexin 1 receptor (OX1R) or orexin 2 receptor (OX2R). This orexin/GPCR-stimulated mTOR activation is sensitive to rapamycin, an inhibitor of mTOR complex 1 (mTORC1) but is independent of two well known mTORC1 activators, Erk and Akt. Rather, our studies indicate that orexin activates mTORC1 via extracellular calcium influx and the lysosome pathway involving v-ATPase and Rag GTPases. Moreover, a cytoplasmic calcium transient is sufficient to mimic orexin/GPCR signaling to mTORC1 activation in a v-ATPase-dependent manner. Together, our studies suggest that the mTORC1 pathway functions downstream of orexin/GPCR signaling, which plays a crucial role in many physiological and metabolic processes.