Organic anion transport study in mutant rats with autosomal recessive conjugated hyperbilirubinemia

The EHBR is a mutant rat strain with congenital conjugated hyperbilirubinemia bred from a Sprague-Dawley rat. Transport of conjugated bilirubin, indocyanine green, and tetrabromosulfophtalein from liver to bile is severely impaired in these rats. Serum bilirubin amounts to 6.0 +/- 0.05 mg/dl (n = 4) in adult rats, with 97% conjugates. The bile flow is reduced to about 65% of the control group, whereas total bile acid in 10-min bile samples is similar. Liver histology of 10 week-old rats revealed neither intracellular pigmentation nor architectural abnormalities.     

Simple and efficient knockdown of His-tagged proteins by ternary molecules consisting of a His-tag ligand,a ubiquitin ligase ligand,and a cell-penetrating peptide

We designed and synthesized hybrid molecules for a protein knockdown method based on the recognition of a His-tag fused to a protein of interest (POI). The synthesized target protein degradation inducers contained three functional moieties: a His-tag ligand (nickel nitrilotriacetic acid [Ni-NTA]), an E3 ligand (bestatin [BS] or MV1), and a carrier peptide (Tat or nonaarginine [R9]). The designed hybrid molecules, BS-Tat-Ni-NTA, MV1-Tat-Ni-NTA, BS-R9-Ni-NTA, and MV1-R9-Ni-NTA, efficiently degraded His-tagged cellular retinoic acid binding protein 2 via the ubiquitin–proteasome system (UPS). This system will become a useful tool for research into selective protein degradation inducers that act via the UPS.     

Development of an ON/OFF switchable fluorescent probe targeting His tag fused proteins in living cells

The fluorescent labeling of target proteins is useful for analyzing their functions and localization in cells, and several fluorescent probes have been developed. However, the fusion of tags such as green fluorescent protein (GFP) to target proteins occasionally affects their functions and/or localization in living cells. Therefore, an imaging method that uses short peptide tags such as hexa-histidine (the His tag) has been attracting increasing attention. Few studies have investigated ON/OFF switchable fluorescent probes for intracellular His-tagged proteins. We herein developed a novel ON/OFF switchable probe for imaging targeted intracellular proteins fused with a CH6 tag, which is composed of one cysteine residue and six histidine residues.     

Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C-terminal alpha helix, modified by a variety of environmental factors as studied by (13)C-NMR.

We have examined the (13)C-NMR spectra of [3-(13)C] Ala-labeled bacteriorhodopsin and its mutants by varying a variety of environmental or intrinsic factors such as ionic strength, temperature, pH, truncation of the C-terminal alpha helix, and site-directed mutation at cytoplasmic loops, in order to gain insight into a plausible surface structure arising from the C-terminal alpha helix and loops. It is found that the surface structure can be characterized as a complex stabilized by salt bridges or metal-mediated linkages among charged side chains. The surface complex in bacteriorhodopsin is most pronounced under the conditions of 10 mM NaCl at neutral pH but is destabilized to yield relaxed states when environmental factors are changed to high ionic strength, low pH and higher temperature. These two states were readily distinguished by associated spectral changes, including suppressed (cross polarization-magic angle spinning NMR) or displaced (upfield) (13)C signals from the C-terminal alpha helix, or modified spectral features in the loop region. It is also noteworthy that such spectral changes, when going from the complexed to relaxed states, occur either when the C-terminal alpha helix is deleted or site-directed mutations were introduced at a cytoplasmic loop. These observations clearly emphasize that organization of the cytoplasmic surface complex is important in the stabilization of the three-dimensional structure at ambient temperature, and subsequently plays an essential role in biological functions.     

Three-Dimensional Imaging of the Intracellular Fate of Plasmid DNA and Transgene Expression: ZsGreen1 and Tissue Clearing Method CUBIC Are an Optimal Combination for Multicolor Deep Imaging in Murine Tissues

Evaluation methods for determining the distribution of transgene expression in the body and the in vivo fate of viral and non-viral vectors are necessary for successful development of in vivo gene delivery systems. Here, we evaluated the spatial distribution of transgene expression using tissue clearing methods. After hydrodynamic injection of plasmid DNA into mice, whole tissues were subjected to tissue clearing. Tissue clearing followed by confocal laser scanning microscopy enabled evaluation of the three-dimensional distribution of transgene expression without preparation of tissue sections. Among the tested clearing methods (Clear^T2, SeeDB, and CUBIC), CUBIC was the most suitable method for determining the spatial distribution of transgene expression in not only the liver but also other tissues such as the kidney and lung. In terms of the type of fluorescent protein, the observable depth for green fluorescent protein ZsGreen1 was slightly greater than that for red fluorescent protein tdTomato. We observed a depth of ~1.5 mm for the liver and 500 μm for other tissues without preparation of tissue sections. Furthermore, we succeeded in multicolor deep imaging of the intracellular fate of plasmid DNA in the murine liver. Thus, tissue clearing would be a powerful approach for determining the spatial distribution of plasmid DNA and transgene expression in various murine tissues.     

Liver- and lobe-selective gene transfection following the instillation of plasmid DNA to the liver surface in mice

The present study has undertaken the liver- and lobe-selective gene transfections following the instillation of plasmid DNA (pDNA) to the liver surface in mice. The luciferase levels produced in the applied (left) liver lobe at 6 h after liver surface instillation of pDNA were significantly higher than those produced in the other tissues assayed, and ranged from 8.5-fold higher in other liver lobes to 320-fold higher in other tissues. After small intestine surface instillation of pDNA, the gene expression was a little detected in the tissues assayed. Following liver surface instillation of pDNA at a time from 2 to 48 h or at a volume from 15 to 120 microl, the gene expressions of the applied liver lobe were always significantly higher than those of other liver lobes and other tissues. We demonstrated the novel liver- and lobe-selective gene transfection utilizing the instillation to the liver surface.     

Cationic liposomes-mediated plasmid DNA delivery in murine hepatitis induced by carbon tetrachloride

In order to elucidate the influence of hepatic disease stage on cationic liposomes-mediated gene delivery, we investigated the cationic liposomes-mediated plasmid DNA delivery with time in murine hepatitis induced by subcutaneous injection of CCl₄. Liver injury after injection of CCl₄ was confirmed by the determination of serum aspartate aminotransferase and alanine aminotransferase activities. Two kinds of liposomes constructed with N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethlylammoniumchloride and dioleylphosphatidylethanolamine (DOTMA-DOPE) or DOTMA and cholesterol (DOTMA-CHOL) were used for the gene-delivery vector. We determined luciferase activities in various organs after the intravenous administration of the lipoplexes. The CCl₄-treated mice administered with DOTMA-DOPE lipoplexes showed the more significant decreases of transgene expression in the liver and spleen at 18 hours after CCl₄ injection. On the other hand, the CCl₄-treated mice administered with DOTMA-CHOL lipoplexes showed a significant increase in the liver at 48 hours. In conclusion, our findings demonstrate that murine hepatitis induced by CCl₄ can influence cationic liposomes-mediated plasmid DNA delivery. The extent of influences was also affected by lipid contents. These results indicate the necessity of considering the timing and the formulation for gene therapy according to the disease stage.     

Shear-Induced Migration of a Transmembrane Protein within a Vesicle

Biomembranes feature phospholipid bilayers and serve as the interface between cells or organelles and the extracellular and/or cellular environment. Lipids can move freely throughout the membrane; the lipid bilayer behaves like a fluid. Such fluidity is important in terms of the actions of membrane transport proteins, which often mediate biological functions; membrane protein motion has attracted a great deal of attention. Because the proteins are small, diffusion phenomena are often in play, but flow-induced transport has rarely been addressed. Here, we used a dissipative particle dynamics approach to investigate flow-induced membrane protein transport. We analyzed the drift of a membrane protein located within a vesicle. Under the influence of shear flow, the protein gradually migrated toward the vorticity axis via a random walk, and the probability of retention around the axis was high. To understand the mechanism of protein migration, we varied both shear strength and protein size. Protein migration was induced by the balance between the drag and thermodynamic diffusion forces and could be represented by the Péclet number. These results improve our understanding of flow-induced membrane protein transport.     

Male Reproductive Success in a Promiscuous Armoured Catfish Corydoras Aeneus (Callichthyidae)

Among a variety of fish mating systems, promiscuity with random-mating seems to be most prevalent. However, detailed studies of promiscuity have been rare due partly to the peculiar difficulty in examination of male mating and reproductive success in the random mating. Females of the armoured catfish Corydoras aeneus (no sexual dimorphism other than size of males > females) spawn 10–20 egg-clutches with multiple males at a time, but an entire egg clutch is inseminated by sperm of a single male. We studied mating system of this fish in aquarium. Males had neither mating territories nor monopolized females, never being aggressive against rival males. Evidence of female preference for certain male traits including size was not detected. Females mated a male in proportion to his relative courtship frequency among males. Courtship frequency was not related to male size, and male mating success was not different between small and large males. Clutch size and insemination rate were different neither between small and large males nor between frequently and less frequently courting males. Thus, the male reproductive success will not be related to the male size, but directly to courtship frequency, indicating the random mating in this fish. There seemed to be fecundity advantage with size in female, and the consequent sexual difference in energy allocation will be responsible to the sexual dimorphism. We also discuss the low male-GSI in this promiscuous fish in which sperm competition hardly occurred.