Cellular uptake and subsequent intracellular trafficking of R8-liposomes introduced at low temperature

Intracellular trafficking is a determining factor in the transgene expression efficiency of gene vectors. In the present study, the mechanism of the cellular uptake of octaarginine (R8)-modified liposomes, when introduced at 37 degrees C and 4 degrees C, was investigated in living cells. Compared with 37 degrees C, the uptake of R8-liposomes was only slightly reduced at 4 degrees C. Dual imaging of liposomes and plasma membranes revealed that R8-liposomes were internalized by vesicular transport, and partially escaped to the cytosol at the perinuclear region at 37 degrees C. When introduced at 4 degrees C, intracellular liposomes were observed within a specific region close to the plasma membrane, and internalization of the plasma membrane was completely inhibited. Therefore, at 4 degrees C, R8-liposomes appear to enter cells via unique pathway, which is separate and distinct from energy-dependent vesicular transport. The subsequent nuclear delivery of encapsulated pDNA, when introduced at 4 degrees C, was less prominent compared with those introduced at 37 degrees C. Collectively, these findings demonstrate that a vesicular transport-independent pathway is responsible for the cellular uptake of liposomes. In addition, the uptake route is closely related to the subsequent nuclear delivery process; the operation of an endogenous vesicular sorting system is advantageous for the nuclear delivery of pDNA.     

Rapid identification of marine bioluminescent bacteria by amplified 16S ribosomal RNA gene restriction analysis

To rapidly identify natural isolates of marine bioluminescent bacteria, we developed amplified ribosomal DNA restriction analysis (ARDRA) methods. ARDRA, which is based on the restriction patterns of 16S rRNA gene digested with five enzymes (EcoRI, DdeI, HhaI, HinfI, RsaI), clearly distinguished the 14 species of marine bioluminescent bacteria currently known, which belong to the genera Vibrio, Photobacterium, and Shewanella. When we applied ARDRA to 129 natural isolates from two cruises in Sagami Bay, Japan, 127 were grouped into six ARDRA types with distinctive restriction patterns; these isolates represented the bioluminescent species, P. angustum, P. leiognathi, P. phosphoreum, S. woodyi, V. fischeri, and V. harveyi. The other two isolates showing unexpected ARDRA patterns turned out to have 16S rRNA gene sequences similar to P. leiognathi and P. phosphoreum. Nevertheless, ARDRA provides a simple and fairly robust means for rapid identification of the natural isolates of marine bioluminescent bacteria, and is therefore useful in studying their diversity.     

High-density Integrated Linkage Map Based on SSR Markers in Soybean

A well-saturated molecular linkage map is a prerequisite for modern plant breeding. Several genetic maps have been developed for soybean with various types of molecular markers. Simple sequence repeats (SSRs) are single-locus markers with high allelic variation and are widely applicable to different genotypes. We have now mapped 1810 SSR or sequence-tagged site markers in one or more of three recombinant inbred populations of soybean (the US cultivar ‘Jack’ × the Japanese cultivar ‘Fukuyutaka’, the Chinese cultivar ‘Peking’ × the Japanese cultivar ‘Akita’, and the Japanese cultivar ‘Misuzudaizu’ × the Chinese breeding line ‘Moshidou Gong 503’) and have aligned these markers with the 20 consensus linkage groups (LGs). The total length of the integrated linkage map was 2442.9 cM, and the average number of molecular markers was 90.5 (range of 70–114) for the 20 LGs. We examined allelic diversity for 1238 of the SSR markers among 23 soybean cultivars or lines and a wild accession. The number of alleles per locus ranged from 2 to 7, with an average of 2.8. Our high-density linkage map should facilitate ongoing and future genomic research such as analysis of quantitative trait loci and positional cloning in addition to marker-assisted selection in soybean breeding.Key words: EST-derived SSR marker, integrated linkage map, microsatellite marker, polymorphism information content     

The receptor-like kinase KLAVIER mediates systemic regulation of nodulation and non-symbiotic shoot development in Lotus japonicus

In legumes, the number of symbiotic root nodules is controlled by long-distance communication between the shoot and the root. Mutants defective in this feedback mechanism exhibit a hypernodulating phenotype. Here, we report the identification of a novel leucine-rich repeat receptor-like kinase (LRR-RLK), KLAVIER (KLV), which mediates the systemic negative regulation of nodulation in Lotus japonicus. In leaf, KLV is predominantly expressed in the vascular tissues, as with another LRR-RLK gene, HAR1, which also regulates nodule number. A double-mutant analysis indicated that KLV and HAR1 function in the same genetic pathway that governs the negative regulation of nodulation. LjCLE-RS1 and LjCLE-RS2 represent potential root-derived mobile signals for the HAR1-mediated systemic regulation of nodulation. Overexpression of LjCLE-RS1 or LjCLE-RS2 did not suppress the hypernodulation phenotype of the klv mutant, indicating that KLV is required and acts downstream of LjCLE-RS1 and LjCLE-RS2. In addition to the role of KLV in symbiosis, complementation tests and expression analyses indicated that KLV plays multiple roles in shoot development, including maintenance of shoot apical meristem, vascular continuity, shoot growth and promotion of flowering. Biochemical analyses using transient expression in Nicotiana benthamiana revealed that KLV has the ability to interact with HAR1 and with itself. Together, these results suggest that the potential KLV-HAR1 receptor complex regulates symbiotic nodule development and that KLV is also a key component in other signal transduction pathways that mediate non-symbiotic shoot development.     

Golgi-localized KIAA0725p regulates membrane trafficking from the Golgi apparatus to the plasma membrane in mammalian cells

Mammals have three members of the intracellular phospholipase A₁ protein family (phosphatidic acid preferring-phospholipase A₁, p125, and KIAA0725p). In this study, we showed that KIAA0725p is localized in the Golgi, and is rapidly cycled between the Golgi and cytosol. Catalytic activity is important for targeting of KIAA0725p to Golgi membranes. RNA interference experiments suggested that KIAA0725p contributes to efficient membrane trafficking from the Golgi apparatus to the plasma membrane, but is not involved in brefeldin A-induced Golgi-to-endoplasmic reticulum retrograde transport.

Structured summary

MINT-8019765: KIAA0725 (uniprotkb:O94830) and Beta-COP (uniprotkb:P53618) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-8019775: KIAA0725 (uniprotkb:O94830) and GM130 (uniprotkb:Q5PXD5) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Structural characteristic of terminal dicarboxylic moiety required for apoptogenic activity of alpha-tocopheryl esters

alpha-Tocopheryl succinate (TS) is known to induce apoptosis in various cells and has attracted attention as a chemotherapeutic agent. Recently, we reported the structural significance of the terminal dicarboxylic moiety for the action of TS [J. Nutr. Sci. Vitaminol. 49 (2003) 310-314]. In this study, to determine details of the relationship between the structure and the function of the terminal ester moiety of alpha-tocopherol (alpha-T), we synthesized four novel esters, alpha-tocopheryl oxalate (TO), alpha-tocopheryl malonate (TM), alpha-tocopheryl pimelate (TP) and alpha-tocopheryl succinate ethyl ester (TSE), and compared their apoptogenic activities with those of TS, alpha-T, gamma-tocopherol (gamma-T) and two commercially available alpha-T derivatives, alpha-tocopheryl nicotinate (TN) and alpha-tocopheryl acetate (TA), in vascular smooth muscle cells and a mouse breast cancer cell line C127I. TO and TM in addition to TS, but not the others, induced apoptosis in both cells. Particularly, TO was the most potent of all alpha-T derivatives used. The addition of exogenous superoxide dismutase (SOD) significantly prevented the apoptosis induced by TM as well as that by TS as reported previously, but did not affect TO-induced apoptosis. These results suggest that O(2)(-) generated exogenously participates in TM-induced apoptosis but not in TO-induced apoptosis. The difference in their apoptotic effects is attributed to structural properties of the terminal dicarboxylic moiety, which has an inflexible plane conformation in TO, while it is highly flexible in TM and TS.     

Temporary membrane distortion of vascular smooth muscle cells is responsible for their apoptosis induced by platelet-activating factor-like oxidized phospholipids and their degradation product, lysophosphatidylcholine

To obtain information about the mechanism of apoptosis induced by oxidized low density lipoproteins (oxLDL) in atherosclerotic plaques, we examined the effects of lysophosphatidylcholine (LPC) and platelet-activating factor (PAF)-like lipids (PAF-LL), which can be derived from oxLDL, on rat vascular smooth muscle cells (VSMC). All the lipids with different structures examined induced apoptosis of VSMC, so we studied the mechanism of induction of apoptosis by LPC. LPC-induced apoptosis was inhibited by alpha-tocopherol (alpha-T) and cholesterol (Chol), but not by other antioxidants such as palmitoyl ascorbic acid and PAF receptor antagonist. The cells temporarily became spherical and highly permeable before induction of apoptosis, and their change in shape was prevented by alpha-T and Chol. From these results, we suggest that the apoptosis induced by oxLDL-derived phospholipids in VSMC is caused by temporary membrane distortion, not through specific receptors.     

Identification of surrogate ligands for orphan G protein-coupled receptors

We prepared fusion proteins with an alpha subunit of G protein Gi (Gi1alpha) of 26 orphan G protein-coupled receptors (GPCRs) and with Gsalpha of 10 orphan GPCRs, most of which had been identified from the human genome previously [FEBS Lett 520 (2002) 97]. Ligands for these fusion proteins were screened from a library consisting of approximately 1000 authentic compounds by measuring their effect on [35S]GTPgammaS binding to membrane preparations of insect Sf9 cells expressing these fusion proteins. Eleven compounds were found to act as surrogate agonists for a GPCR-Gsalpha and four GPCR-Gialpha fusion proteins, a compound as an inverse agonist for two GPCR-Gsalpha fusion proteins, and a compound as an endogenous agonist for a GPCR-Gialpha fusion protein.     

Identification of radiation-sensitive mutants in the Medaka, Oryzias latipes

We screened populations of N-ethyl-N-nitrosourea (ENU)-mutagenized Medaka, (Oryzias latipes) for radiation-sensitive mutants to investigate the mechanism of genome stability induced by ionizing radiation in developing embryos. F3 embryos derived from male founders that were homozygous for induced the mutations were irradiated with gamma-rays at the organogenesis stage (48hpf) at a dose that did not cause malformation in wild-type embryos. We screened 2130 F2 pairs and identified three types of mutants with high incidence of radiation-induced curly tailed (ric) malformations using a low dose of irradiation. The homozygous strain from one of these mutants, ric1, which is highly fertile and easy to breed, was established and characterized related to gamma-irradiation response. The ric1 strain also showed higher incidence of malformation and lower hatchability compared to the wild-type CAB strain after gamma-irradiation at the morula and pre-early gastrula stages. We found that the decrease in hatching success after gamma-irradiation, depends on the maternal genotype at the ric1 locus. Terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling assays showed a high frequency of apoptosis in the ric1 embryos immediately after gamma-irradiation at the pre-early gastrula stage but apoptotic cells were not observed before midblastula transition (MBT). The neutral comet assay revealed that the ric1 mutant has a defect in the rapid repair of DNA double-strand breaks induced by gamma-rays. These results suggest that RIC1 is involved in the DNA double strand break repair in embryos from morula to organogenesis stages, and unrepaired DNA double strand breaks in ric1 trigger apoptosis after MBT. These results support the use of the ric1 strain for investigating various biological consequences of DNA double strand breaks in vivo and for sensitive monitoring of genotoxicity related to low dose radiation.