MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion

Mitochondria are the principal producers of energy in higher cells. Mitochondrial dysfunction is implicated in a variety of human diseases, including cancer and neurodegenerative disorders. Effective medical therapies for such diseases will ultimately require targeted delivery of therapeutic proteins or nucleic acids to the mitochondria, which will be achieved through innovations in the nanotechnology of intracellular trafficking. Here we describe a liposome-based carrier that delivers its macromolecular cargo to the mitochondrial interior via membrane fusion. These liposome particles, which we call MITO-Porters, carry octaarginine surface modifications to stimulate their entry into cells as intact vesicles (via macropinocytosis). We identified lipid compositions for the MITO-Porter which promote both its fusion with the mitochondrial membrane and the release of its cargo to the intra-mitochondrial compartment in living cells. Thus, the MITO-Porter holds promise as an efficacious system for the delivery of both large and small therapeutic molecules into mitochondria.     

Recombinant human thioredoxin suppresses lipopolysaccharide-induced bronchoalveolar neutrophil infiltration in rat

Human thioredoxin (TRX) is a multifunctional redox-active protein. We previously reported that the intraperitoneal administration of recombinant human thioredoxin (rhTRX) attenuates inflammatory cytokine- or bleomycin-induced lung injury in mice. In this study, the effect of rhTRX injected intravenously after lipopolysaccharide (LPS) injection was analyzed in rats. Rats were injected with LPS followed by treatment with rhTRX. Although the bolus injection exerted no protective effect, continuous intravenous administration of rhTRX significantly suppressed percentage number of neutrophils in bronchoalveolar lavage fluid. Histological examination also showed that rhTRX decreased neutrophil infiltration in the lung tissues. Administered rhTRX was mainly excreted into the urine and the tissue accumulation of rhTRX in the lung was marginal. LPS-induced oxidative stress in the lung was slight in this model. These results demonstrated that continuous intravenous administration of rhTRX suppresses LPS-induced bronchoalveolar neutrophil infiltration by an anti-chemotactic effect. Administration of rhTRX did not promote the tumor growth nor affect chemosensitivity in the xenotransplantation model, suggesting the safety of rhTRX therapy for cancer patients.     

The occurrence of two genotypes of the planktonic foraminifer Globigerinoides ruber (white) and paleo-environmental implications

Planktonic foraminifera provide a record of the upper ocean environment in the chemical and isotopic composition of individual shells. Globigerinoides ruber is a common tropical–subtropical planktonic foraminifer, and this species is used extensively for reconstruction of the paleo-environment. The different stable isotopic compositions of two morphotypes, G. ruber sensu stricto (s.s.) and G. ruber sensu lato (s.l.), first identified in sediments, suggested that G. ruber s.s. was dwelling in the upper 30 m of the water column and G. ruber s.l. at greater depths. Plankton tows and sediment trap experiments provided additional evidence distinguishing the two morphotypes and their habitats and invited the question as to whether the two morphotypes could be distinguished genetically. In this study, using phylogenetic analysis of nuclear partial small subunit ribosomal DNA (SSU rDNA) gene sequences representing 12 new and 16 known sequences, we identified four genotypes within G. ruber white variation; one of which is a sister group of Globigerinoides conglobatus, whereas the three others were sister groups of the G. ruber pink variation. Moreover, these two major groups corresponded to morphological differences described as G. ruber s.l. and s.s., respectively. This genetic evidence corroborates differences between the two morphotypes in the isotope record, and it will contribute to a more precise reconstruction of the thermal structure of the water column.     

Analysis of hunger-driven gene expression in the Drosophila melanogaster larval central nervous system

A transposon-inserted mutant of Drosophila melanogaster was recently identified, and the larvae show no food preference (Ryuda and Hayakawa, 2005). To reveal the genetic mechanism underlying the preference change in this mutant, a large-scale oligo-DNA microarray screening was carried out to identify genes whose expression is different in control and mutant strains. We focused especially on hunger-driven changes in gene expression in the larval central nervous system (CNS) of both strains, because the state of food depletion should promote a feeding response due to changed expression of certain genes in the CNS. We identified 22 genes whose expression changed after starvation in either or both of the two strains. Quantitative RT-PCR analyses confirmed the expression changes in four genes, CG6271, CG6277, CG7953, and new glue 3 (ng3, encoding a putative structural molecule). CG6271 and CG6277 encode triacylglycerol lipase, and CG7953 produces a protein homologous to a juvenile hormone (JH) binding protein. The expression of these two groups of genes was enhanced in control strain larvae with a normal food preference but not in GS1189 strain larvae. Given that these genes contribute to mediating hunger-driven changes in food preference and intake in D. melanogaster larvae, the dysfunction of these key genes could cause the defect in food preference observed in GS1189-strain larvae.     

Abundance and pigment type composition of picocyanobacteria in Barguzin Bay, Lake Baikal

In Lake Baikal, picocyanobacteria are the most important primary producers during the summer. Freshwater picocyanobacteria are discriminated into either the phycoerythrin (PE)-rich or the phycocyanin (PC)-rich types according to their pigment composition. The distributions of these two types of picocyanobacteria were investigated in Barguzin Bay. The PC-rich type accounted for >98% of the total picocyanobacteria at the station near the shore of the bay where river water flows directly in. In the offshore area of the lake, all of the picocyanobacteria cells were of the PE-rich type. In addition, the occurrence of the PC-rich type was restricted to the station, where the attenuation coefficient exceeded 0.25 m⁻¹. Near the shore, where the turbidity was high (>1 NTU), the cell densities of both the PE- and PC-rich types increased away from the river mouth. This indicates that the PC-rich type cells grow near the shore of the bay where turbidity is high. Since the PC-rich type could not grow well when cells were incubated in offshore lake water, restricted distribution of the PC-rich type could also be explained by their growth capability. The present study clearly demonstrated the shift in the pigment type composition of picocyanobacteria from the coastal to the pelagic zone of Lake Baikal. The co-existence of the two pigment types probably enables the abundance of the picocyanobacterial community to be stable over a broader range of environmental conditions than would be possible for a single pigment type.     

Genomic analysis of immunity in a Urochordate and the emergence of the vertebrate immune system: “waiting for Godot”

Genome-wide sequence analysis in the invertebrate chordate, Ciona intestinalis, has provided a comprehensive picture of immune-related genes in an organism that occupies a key phylogenetic position in vertebrate evolution. The pivotal genes for adaptive immunity, such as the major histocompatibility complex (MHC) class I and II genes, T-cell receptors, or dimeric immunoglobulin molecules, have not been identified in the Ciona genome. Many genes involved in innate immunity have been identified, including complement components, Toll-like receptors, and the genes involved in intracellular signal transduction of immune responses, and show both expansion and unexpected diversity in comparison with the vertebrates. In addition, a number of genes were identified which predicted integral membrane proteins with extracellular C-type lectin or immunoglobulin domains and intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based activation motifs (ITAMs) (plus their associated signal transduction molecules), suggesting that activating and inhibitory receptors have an MHC-independent function and an early evolutionary origin. A crucial component of vertebrate adaptive immunity is somatic diversification, and the recombination activating genes (RAG) and activation-induced cytidine deaminase (AID) genes responsible for the Generation of diversity are not present in Ciona. However, there are key V regions, the essential feature of an immunoglobulin superfamily VC1-like core, and possible proto-MHC regions scattered throughout the genome waiting for Godot.     

Lats2 is an essential mitotic regulator required for the coordination of cell division

Tumor suppressor Lats2 is a member of the conserved Dbf2 kinase family. It localizes to the centrosome and has been implicated in regulation of the cell cycle and apoptosis. However, the in vivo function of this kinase remains unclear. Here, we show that complete disruption of the gene encoding Lats2 in mice causes developmental defects in the nervous system and embryonic lethality. Furthermore, mutant cells derived from total LATS2-knock-out embryos exhibit mitotic defects including centrosome fragmentation and cytokinesis defects, followed by nuclear enlargement and multinucleation. We show that the Mob1 family, a regulator of mitotic exit, associates with Lats2 to induce its activation. We also show that the complete LATS2-knock-out cells exhibit an acceleration of exit from mitosis and marked down-regulation of critical mitotic regulators. These results suggest that Lats2 plays an essential mitotic role in coordinating accurate cytokinesis completion, governing the stabilization of other mitotic regulators.     

Temperature-controlled atmospheric-pressure plasma treatment induces protein uptake via clathrin-mediated endocytosis in tobacco cells

Previously, we developed a method that uses temperature-controlled atmospheric-pressure plasma to induce protein uptake in plant cells. In the present work, we examined the mechanism underlying such uptake of a fluorescent-tagged protein in tobacco leaf cells. Intact leaf tissue was irradiated with N₂ plasma generated by a multi-gas plasma jet and then exposed to the test protein (histidine-tagged superfolder green fluorescence protein fused to adenylate cyclase); fluorescence intensity was then monitored over time as an index of protein uptake. Confocal microscopy revealed that protein uptake potential was retained in the leaf tissue for at least 3 h after plasma treatment. Further examination indicated that the introduced protein reached a similar amount to that after overnight incubation at approximately 5 h after irradiation. Inhibitor experiments revealed that protein uptake was significantly suppressed compared with negative controls by pretreatment with sodium azide (inhibitor of adenosine triphosphate hydrolysis) or sucrose or brefeldin A (inhibitors of clathrin-mediated endocytosis) but not by pretreatment with genistein (inhibitor of caveolae/raft-mediated endocytosis) or cytochalasin D (inhibitor of micropinocytosis/phagocytosis), indicating that the N₂ plasma treatment induced protein transportation across the plant plasma membrane via clathrin-mediated endocytosis.