Akitoyo Ichinose

Binding of Cationic α-Helical Peptides to Plasmid DNA and Their Gene Transfer Abilities into Cells

Polycationic reagents such as cationic lipids and poly-l-lysine are widely used for gene transfer into cells in vitro and show promise as vectors for in vivo gene therapy applications as nonviral gene transfer techniques. We have developed a novel transfection method using cationic amphiphilic α-helical oligopeptides with repeated sequences. Oligopeptide has the advantages of being easily designed and modified because of its simple structure. In this study, we synthesized five kinds of peptides of which the total chain length and the width of the hydrophobic region were changed. The binding of the peptides to plasmid DNA was evaluated by agarose gel electrophoresis. It was found that the long and/or hydrophobic peptides can strongly bind to the DNA. The formation of large aggregates with a 0.5–5-μm diameter, which consisted of the long peptides and the DNA, was observed by electron microscopy. The transfection abilities of the peptides were determined by the expression of luciferase from its cDNA in COS-7 cells. The long peptides showed high transfection abilities. As a result, it could be said that the transfection ability of these peptides was parallel to their ability to form aggregates with DNA. Furthermore, the transfection ability was increased by the addition of chloroquine in the transfection procedure. This result indicated that the internalization of the peptide-DNA aggregates would be mediated by the endocytosis pathway.

Discovery of the First Insect Nidovirus, a Missing Evolutionary Link in the Emergence of the Largest RNA Virus Genomes

Nidoviruses with large genomes (26.3–31.7 kb; ‘large nidoviruses’), including Coronaviridae and Roniviridae, are the most complex positive-sense single-stranded RNA (ssRNA+) viruses. Based on genome size, they are far separated from all other ssRNA+ viruses (below 19.6 kb), including the distantly related Arteriviridae (12.7–15.7 kb; ‘small nidoviruses’). Exceptionally for ssRNA+ viruses, large nidoviruses encode a 3′-5′exoribonuclease (ExoN) that was implicated in controlling RNA replication fidelity. Its acquisition may have given rise to the ancestor of large nidoviruses, a hypothesis for which we here provide evolutionary support using comparative genomics involving the newly discovered first insect-borne nidovirus. This Nam Dinh virus (NDiV), named after a Vietnamese province, was isolated from mosquitoes and is yet to be linked to any pathology. The genome of this enveloped 60–80 nm virus is 20,192 nt and has a nidovirus-like polycistronic organization including two large, partially overlapping open reading frames (ORF) 1a and 1b followed by several smaller 3′-proximal ORFs. Peptide sequencing assigned three virion proteins to ORFs 2a, 2b, and 3, which are expressed from two 3′-coterminal subgenomic RNAs. The NDiV ORF1a/ORF1b frameshifting signal and various replicative proteins were tentatively mapped to canonical positions in the nidovirus genome. They include six nidovirus-wide conserved replicase domains, as well as the ExoN and 2′-O-methyltransferase that are specific to large nidoviruses. NDiV ORF1b also encodes a putative N7-methyltransferase, identified in a subset of large nidoviruses, but not the uridylate-specific endonuclease that – in deviation from the current paradigm - is present exclusively in the currently known vertebrate nidoviruses. Rooted phylogenetic inference by Bayesian and Maximum Likelihood methods indicates that NDiV clusters with roniviruses and that its branch diverged from large nidoviruses early after they split from small nidoviruses. Together these characteristics identify NDiV as the prototype of a new nidovirus family and a missing link in the transition from small to large nidoviruses.

Intracellular visualization of tetrodotoxin (TTX) in the skin of a puffer Tetraodon nigroviridis by immunoenzymatic technique

Micro-distributions of tetrodotoxin (TTX) in the skin of a brackish-water puffer Tetraodon nigroviridis were investigated by means of a monoclonal anti-TTX antibody under light and transmission electron microscope. In light microscopy TTX antigen was visualized as brown color in undifferentiated basal cells and succiform cells of the skin, while in electron microscopy TTX was detected as black dots of immunogold in lysosomes of basal cells. From the results, it can be inferred that when TTX from the blood plasma of T. nigroviridis enters into the undifferentiated basal cells through diffusion, it is taken to the lysosomes by phagocytosis and exists there through binding with internal constituent(s) of the organelles.

Tanay virus, a new species of virus isolated from mosquitoes in the Philippines.

In 2005, we isolated a new species of virus from mosquitoes in the Philippines. The virion was elliptical in shape and had a short single projection. The virus was named Tanay virus (TANAV) after the locality in which it was found. TANAV genomic RNA was a 9562 nt+poly-A positive strand, and polycistronic. The longest ORF contained putative RNA-dependent RNA polymerase (RdRP); however, conserved short motifs in the RdRP were permuted. TANAV was phylogenetically close to Negevirus, a recently proposed taxon of viruses isolated from haemophagic insects, and to some plant viruses, such as citrus leprosis virus C, hibiscus green spot virus and blueberry necrotic ring blotch virus. In this paper, we describe TANAV and the permuted structure of its RdRP, and discuss its phylogeny together with those of plant viruses and negevirus.

Possible Presence of a Capsule in Branhamella catarrhalis

Clinical isolates of Branhamella catarrhalis from patients with respiratory infections were used in this study. Electron microscopic observation after treating Branhamella catarrhalis with immune serum and ruthenium red revealed the capsule. In the phagocytosis test, most organisms were not ingested by human polymorphonuclear neutrophils in the presence of normal rabbit serum (NRS), while organisms were primarily cell associated and apparently ingested in the presence of immunized rabbit serum (IRS). The capsule may be one of the virulence factors in this bacteria. This study demonstrates the possible presence of a capsule in Branhamella catarrhalis.

Direct binding of gangliosides to Helicobacter pylori vacuolating cytotoxin (VacA) neutralizes its toxin activity

Gangliosides are target receptors for bacterial entry, yet those present in human milk exhibit a protective role against bacterial infection. Here, we show that treatment with ganglioside mixture at a concentration of 100 microg/mL resulted in significant inhibition of the vacuole formation activity of Helicobacter pylori vacuolating cytotoxin (VacA) in gastric epithelial cancer AZ-521 cells. All gangliosides (GM1, GM2, GM3, GD1a, GD1b, GD3 and GT1b) examined showed good neutralizing capacity against VacA. A pull-down assay was performed using lyso-GM1 coupled to Sepharose as the tagged polysaccharide polymer to capture VacA from H. pylori culture supernatant. GM1-VacA complexes were successfully precipitated, suggesting that GM1 binds directly to VacA. The hydrodynamic binding of lyso-GM1 and VacA measured by fluorescence correlation spectroscopy had a K(d) value of 190 nM. VacA also bound to lyso-GM1 at pH 2 corresponding to the physiological pH of human stomach. Collectively, these results showed that direct binding of H. pylori VacA to free gangliosides neutralizes the toxin activity of VacA. These findings offer an alternative insight into the role of gangliosides in VacA toxicity and the pathogenesis of H. pylori.

Role of lipooligosaccharide in the attachment of Moraxella catarrhalis to human pharyngeal epithelial cells

The goal of this study was to determine the role of lipooligosaccharide in the attachment of Moraxella catarrhalis to human pharyngeal epithelial cells. Strain 2951 and its Pk mutant strain 2951 galE were used in this study. This study suggests that the Pk epitope of LOS is not an adhesin for M. catarrhalis, but plays a crucial role by its surface charge in the initial stage of attachment.

Expression of fimbriae and host response in Branhamella catarrhalis respiratory infections

Sputum during the acute exacerbation of chronic respiratory diseases were observed under the electron microscope, to determine the in vivo expression of surface structures of Branhamella catarrhalis (B. catarrhalis), the polymorphonuclear neutrophil (PMN) response to B. catarrhalis infections, and the composition of sputum. It was found that during infection fimbriae are expressed in B. catarrhalis. However, there were sparsely to densely fimbriated bacteria in each sputum sample. The length of the fimbriae were from 50 to 76 nm. In the sparsely fimbriated B. catarrhalis, external to the cell wall, a thin, granular, electron‐dense layer was observed. Due to the presence of fimbriae, this layer was not seen in densely fimbriated B. catarrhalis. Blebs were also found in B. catarrhalis. PMNs were found to phagocytose both B. catarrhalis and debris. Evidence was found that debris were formed mainly by the destruction of PMNs. Bacteria as well as debris were phagocytosed by PMNs.

In vitro cultivation and electron microscopy characterization of Trachipleistophora anthropophthera isolated from the cornea of an AIDS patient

Abstract. We describe an in vitro culture technique for a microsporidian isolated from the corneal biopsy of an HIV‐infected patient. The corneal biopsy was inoculated into a monolayer culture of fibroblasts derived from newborn mouse brain and incubated at 37°C in an atmosphere of 5% CO2. Minimum essential medium supplemented with 2% fetal bovine serum appeared to be an optimum medium for growth and maintenance of the parasite and for production of large numbers of spores. This microsporidian was identified as Trachipleistophora anthropophthera based on ultrastructural features. It forms two types of sporophorous vesicles and two types of spores simultaneously: polysporous vesicle type I with eight or more oval spores, 3.7–4.0 μm by 2.0–2.3 μm, and bisporous vesicle type II with two round spores, 1.7–2.2 μm by 1.6–2.0 μm in size.

Alarin but not its alternative-splicing form, GALP (Galanin-like peptide) has antimicrobial activity.

Alarin is an alternative-splicing form of GALP (galanin-like peptide). It shares only 5 conserved amino acids at the N-terminal region with GALP which is involved in a diverse range of normal brain functions. This study seeks to investigate whether alarin has additional functions due to its differences from GALP. Here, we have shown using a radial diffusion assay that alarin but not GALP inhibited the growth of Escherichia coli (strain ML-35). The conserved N-terminal region, however, remained essential for the antimicrobial activity of alarin as truncated peptides showed reduced killing effect. Moreover, alarin inhibited the growth of E. coli in a similar potency as human cathelicidin LL-37, a well-studied antimicrobial peptide. Electron microscopy further showed that alarin induced bacterial membrane blebbing but unlike LL-37, it did not cause hemolysis of erythrocytes. In addition, alarin is only active against the gram-negative bacteria, E. coli but not the gram-positive bacteria, Staphylococcus aureus. Thus, these data suggest that alarin has potentials as an antimicrobial and should be considered for the development in human therapeutics.