Impact of liver-targeted hydrodynamic gene delivery on hepatic microcirculation.

<p>The total blood volume (IHB, white square) and the blood oxygenation (ISO₂, black diamond) in regional hepatic tissue under normal conditions (<b>a</b>), prior to, during, and after the liver-targeted hydrodynamic saline injection in 10 sec (<b>b</b>), 15 sec (<b>c</b>), and 20 sec (<b>d</b>) to rat livers.</p

Liver-targeted hydrodynamic gene delivery.

<p>(<b>a</b>) Location of the balloon catheter in the hepatic vein. A small amount of contrast medium was injected into the vein to confirm the occlusion of the blood flow and distribution of injected solution during a liver lobe-specific hydrodynamic gene delivery. Black arrows represent the distribution of the contrast medium in the injected liver lobe. (<b>b</b>) Immunohistochemical staining of the liver. Liver samples from control and plasmid DNA injected liver were stained with anti-luciferase antibodies. Scale bar represents 50 µm. White arrow indicates the positively stained hepatocytes. (<b>c</b>) The change of body weight before, after the arrival of the animal to the animal facility. White, gray, and black arrowheads point the hydrodynamic injections in representative 4 dogs. (<b>d–g</b>) Physiological impacts of the sequential, liver-targeted hydrodynamic gene delivery in representative 4 dogs. Physiological parameters before, during, and after the sequential liver-targeted hydrodynamic injection of saline (<b>d</b>), pCMV-Luc (<b>e</b>), pCAG-hAAT (<b>f</b>), or pBS-HCRHP-FIXIA (<b>g</b>). HR, heart rate (white square); SBP, systolic blood pressure (white circle); DBP, diastolic blood pressure (black circle); SpO₂, oxygen saturation (black diamond); BT, body temperature (black square). Black arrows indicate each of the 4 lobe injections.</p

Impact of the procedure on serum cytokine levels.

<p>Blood samples were collected from the cephalic or saphenous veins of dogs before (time  = 0), and 2, 24, 48, 96 h after the first hydrodynamic injection of saline (white diamond), pCAG-hAAT (white square), pBS-HCRHP-FIXIA (white triangle), pCMV-Luc (black cross) or slow drip infusion of the same volume of saline from peripheral vein within 1 h (white circle) or 2 h (black asterisk). Concentrations of TNF-α (<b>a</b>), IL-10 (<b>b</b>), MCP-1 (<b>c</b>), Canine KC (<b>d</b>), IL-6 (<b>e</b>), IFN-γ (<b>f</b>), IL-8 (<b>g</b>), IL-18 (<b>h</b>), IL-4 (<b>i</b>). The values represent mean (n = 2 for each group).</p

Impact of the procedure on serum biochemistry.

<p>Blood samples were collected from the cephalic or saphenous veins of dogs before (time  = 0), and 2, 4, 24, 96 h after the first hydrodynamic injection of saline (white diamond), pCAG-hAAT (white square), pBS-HCRHP-FIXIA (white triangle), pCMV-Luc (black cross) or slow drip infusion of the same volume of saline from peripheral vein within 1 h (white circle) or 2 h (black asterisk). Concentrations of aspartate aminotransferase (AST) (<b>a</b>), alanine aminotransferase (ALT) (<b>b</b>), lactate dehydrogenase (LDH) (<b>c</b>), creatinine (Cre) (<b>d</b>), albumin (Alb) (<b>e</b>), and hematocrit value (<b>f</b>). The values represent mean for hydrodynamic injection groups (n = 2 for each group).</p

pH-Dependent Assembly and Segregation of the Coiled-Coil Segments of Yeast Putative Cargo Receptors Emp46p and Emp47p

<div><p>Emp46p and Emp47p are yeast putative cargo receptors that recycle between the endoplasmic reticulum and the Golgi apparatus. These receptors can form complexes in a pH-dependent manner, but their molecular mechanisms remain unclear. Here, we successfully reproduced their interactions <i>in vitro</i> solely with their coiled-coil segments, which form stable heterotetramers in the neutral condition but segregate at lower pH. Mutational data identified a key glutamate residue of Emp46p that serves as the pH-sensing switch of their oligomer formation. Our findings elucidate the mechanisms of the dynamic cargo receptor interactions in the secretory pathway and the design framework of the environment-responsive molecular assembly and disassembly systems.</p></div

Schematic of the coiled-coil segments of Emp46p^CC and Emp47p^CC.

<p>Residues consisting of a hydrophobic core are represented. Hydrophobic residues are represented by yellow, whereas Glu303 is represented with a red circle.</p

Characterization of oligomeric states of Emp46p^CC and Emp47p^CC.

<p>(A) CD spectra of Emp46p^CC (orange) and Emp47p^CC (green). (B) Gel filtration profiles of Emp46p^CC (orange) and Emp47p^CC (green) at pH 7.4. (C) Distribution of sedimentation coefficients derived from sedimentation velocity analytical ultracentrifugation (SV-AUC) experiments. The orange line indicates Emp46p^CC (75 μM), whereas the green line indicates Emp47p^CC (75 μM). (D) Mass spectra of Emp46p^CC (top) and Emp47p^CC (bottom) at pH 7.4. Peaks corresponding to the monomer and dimer of Emp46p^CC are indicated by purple and red dots with charge states, respectively. Peaks corresponding to the monomer and tetramer of Emp47p^CC are indicated by blue and green dots with charge states, respectively.</p

Characterization of complex formation between Emp46p^CC and Emp47p^CC at pH 7.4.

<p>(A) Gel-filtration profiles of the mixture of Emp46p^CC and Emp47p^CC at 1:0, 1:0.5, 1:1, 1:1.5, and 1:2 molar ratios. White and black arrows indicate the elution positions of Emp46p^CC alone and oligomers involving Emp47p^CC (and Emp46p^CC), respectively. (B) Mass spectra of the mixtures of Emp46p^CC and Emp47p^CC at pH 7.4. The concentration of Emp47p^CC was fixed and that of Emp46p^CC was varied at the molar ratio indicated. Peaks corresponding to 1:3 and 2:2 heterotetramers of Emp46p^CC and Emp47p^CC are indicated by magenta and cyan dots with charge states, respectively. Peaks corresponding to the homotetramer of Emp47p^CC are indicated by a green dot with charge states.</p

Representative photomicrographs showing the effects of CSAA and CDAA diet on liver histology in rats.

<p>Frozen liver tissues were stained with Oil Red O (A). Rats were fed CSAA diet and CDAA diet, co-administered with saline, or CSAA diet and CDAA diet, co-administered with nicotine, for 6 weeks. Original magnification, ×200. Quantitative analysis of changes in Oil Red O positive areas in respective groups (B). Data are expressed as means ± SE of six to eight rats. (* p < 0.05, ** p < 0.01 compared with the respective group).</p

Representative photomicrographs showing the effects of hepatic branch vagotomy and sham operation on CDAA-diet-induced hepatic steatosis in rats.

<p>Frozen liver sections were stained with Oil Red O (A). Quantitative analysis of changes in Oil Red O positive lesions in the respective groups (B). Original magnification, ×100. Data are expressed as means ± SE of six rats. (^a p < 0.05 compared with the respective groups).</p