In Vivo Canine Model Comparison of Cardiovascular Effects of Antidepressants Milnacipran and Imipramine

Milnacipran is a specific serotonin and norepinephrine reuptake inhibitor, which has been widely used against major depressive episodes. In this study, cardiovascular effects of milnacipran were assessed in comparison with those of a typical tricyclic antidepressant imipramine using the halothane-anesthetized dogs. Milnacipran (n = 6) or imipramine (n = 6) was intravenously administrated in three escalating doses of 0.1, 1 and 10 mg/kg over 10 min with a pause of 20 min between the doses. Clinically relevant plasma concentrations were obtained after 0.1–1 mg/kg of milnacipran in this study, whereas therapeutic dose and plasma concentration of imipramine were reported to be similar to those of milnacipran. The low and middle doses of milnacipran hardly affected cardiohemodynamic or electrophysiological variables except that they slightly increased vascular tone and ventricular contraction, whereas same doses of imipramine delayed repolarization process without affecting the other variables. The high dose of both milnacipran and imipramine induced similar extent of negative chronotropic, inotropic and dromotropic effects together with vasoconstriction and repolarization delay. Thus, the effects of milnacipran may be more selective for cardiohemodynamics than for repolarization delay, whereas reverse will be true for imipramine, supporting lack of clinical report of patients with milnacipran-induced long QT syndrome unlike imipramine.     

Development of sugar-based materials for biological devices

Recent progresses of our group dealing with newly developed sugar-based ligands and their metal complexes have been reviewed. Carbohydrate moiety affects on the stereochemistry around the metal center and controls the biological activity of the complex.     

Covalent binding of nitroso-sulfonamides to glutathione S-transferase in guinea pigs with delayed type hypersensitivity

Drug induced allergies are believed to be induced by conjugates consisting of biological macromolecules and active metabolites. The present study investigated whether guinea pig glutathione S-transferase (gpGST), a protein that binds with sulfanilamide (SA) and sulfamethoxazole (SMX), could be detected in the liver cytosol fraction of guinea pigs that intraperitoneally received SA or SMX, and whether gpGST is a carrier protein. We synthesized three nitroso compounds, i.e., 4-nitroso-sulfanilamide (SA-NO), 4-nitrososulfamethoxazole (SMX-NO) and fluorescent-labeled nitroso compound (DNSBA-NO), and examined binding quantities of nitroso compounds to gpGST purified from untreated female guinea pigs. Furthermore, the concentrations of IgG in serum antibody for nitroso compounds were estimated using ELISA. When guinea pigs were sensitized using the three nitroso compounds, the dose dependent skin reactions were confirmed with each compound. In addition, sensitized guinea pigs using each nitroso compound showed positive skin reactions at an elicitation test performed using gpGST alone. The results confirmed synthesis of antibody against gpGST due to hapten sensitization. Therefore, when a nitroso compound binds with gpGST in the body of guinea pigs, nitroso-gpGST acts as a neoantigen, which induces synthesis of autoantibody. Thus, gpGST appears to be one of the carrier proteins that induce sulfa drug-induced allergies. Immunization of guinea pigs with active metabolite of drugs may give information for predicting the occurrence of delayed type hypersensitivity in human.     

Irreversible optical clearing of sclera by dehydration and cross-linking

This study manipulates both clear cornea and opaque sclera by two dehydration processes for revealing the relationship between altered tissue structures and change in optical functions. In contrast to the high levels of light scattering in dehydrated tissues by critical point dry, a simple dehydration at 4-8 °C effectively and significantly improved their visible-light transmission, even in the sclera, with accompanying dense fiber packing. Further improvement in visible-light transmission, from 40-50% to 80-90%, has been achieved by flatting tissue surface with cover glasses during dehydration at low temperature. Such optical clearing of sclera by dehydration is reversible. However, chemical cross-linking effectively stabilizes their densely packed microscopic structures and visible-light transmission at over 50% irreversibly, even at wet conditions. Interestingly, the repetition of both low temperature dehydration/cross-linking treatments effectively reduced the required amounts of cross-linking reagents to keep a high transparency. Wet transparent cross-linked sclera can also show a characteristic strong tensile strength. Furthermore, rabbit corneal epithelium has regenerated on the transparent sclera with cross-linking in vitro.     

Transparent, tough collagen laminates prepared by oriented flow casting, multi-cyclic vitrification and chemical cross-linking

The lamellar architecture found in many natural fibrous tissues has a significant bearing on their specific functions. However, current engineered tissues have simultaneously no realistic structures and no adequate functions. This study demonstrates a two-step process for obtaining structurally mimicking laminates in natural fibrous tissues with good optical and mechanical characters from purified-clinically-safe collagen molecules. Stacked lamella structures can be created by repeating flow casting, with the controlling parallel/orthogonal directionalities of each thin single-layer (2-5 μm in thickness). The transparency of laminates is successfully improved by a unique multi-cyclic vitrification with chemical cross-linking. The directionalities of optical and mechanical functions in laminates are strongly related with the preferential collagen alignments in the laminates. The tensile strength of laminates is extremely higher than any other engineered materials as well as native cornea, which exhibit an orthogonal laminated collagen structure and a good optical transmission.     

Cells immobilized on patterns printed in DNA by an inkjet printer

The ability to two-dimensionally align various kinds of cells freely onto substrate would be a useful tool for analysis of cell-cell interactions. In this study, we aimed to establish a method for attaching cells to the substrate, in which the pattern is drawn by an inkjet printer. Poly-deoxyribonucleic acid (DNA) was immobilized onto the cell surface by use of DNA-conjugated poly(ethylene) glycol-phospholipid (DNA-PEG-lipid), which is the amphiphilic conjugate of PEG-lipid and single-stranded DNA. The surface of the substrate was then modified with the complementary DNA using an inkjet printer. Finally, DNA-immobilized cells were attached onto the substrate through DNA hybridization. The use of the inkjet printer enabled us to draw the DNA pattern accurately on the substrate with a resolution of a few hundred micrometers. DNA-immobilized cells could be attached precisely along the DNA pattern on the substrate. In addition, various kinds of cells could be attached simultaneously by using various sequences of DNA. Our technique is promising for analysis of cell-cell interactions and differentiation induction in stem cell research.     

Prevalence of amino acid mutation in hepatitis C virus core region among Japanese volunteer blood donors

It is not known whether there is a trend of increasing or decreasing incidence of new hepatitis C virus (HCV) infections in Japan. From the treatment point of view, it is important to verify HCV genotypes and the prevalence of treatment-resistant clones of HCV. At the Japanese Red Cross blood centers, all blood samples obtained from blood donation have been screened using serological methods and the minipool nucleic acid amplification testing. One hundred and fourteen donors have been identified over the past 10 years to be HCV RNA-only positive without detectable anti-HCV and were considered to be in the acute phase of HCV infection. There was a trend of decreasing incidence of such new infections among the blood donors. HCV RNA-only-positive samples were examined further for genotyping and HCV RNA quantitation. Genotype 2 (2a plus 2b) was predominant (78.2%) among them followed by genotype 1b (21.2%). Direct sequencing was carried out to detect the possible treatment-resistant mutant clones 70Q and 91M, clones with amino acid substitutions at positions 70 and 91 of the HCV core protein, respectively. 70Q and 91M were found regularly in donors with genotype 1b, but not in those with other genotypes. No particular endemic areas for the mutant clones were identified. There was no significant difference in the mean viral titer between the 70Q mutant type and the non-70Q wild-type. Even in newly infected people, the mutant clone 70Q was detected frequently.     

Thoracotomy reduces intrinsic brain movement caused by heartbeat and respiration: a simple method to prevent motion artifact for in vivo experiments

Recent technical advances in electrophysiological recording and functional imaging from the brain of living animals have promoted our understandings of the brain function, but these in vivo experiments are still technically demanding and often suffer from spontaneous pulsation, i.e., brain movements caused by respiration and heartbeat. Here we report that thoracotomy suppresses the motion artifact to a practically negligible level. This simple method will be useful in a wide variety of in vivo experiments, such as patch-clamp physiology, and optical imaging of neurons, glial cell, and blood vessels.     

Attenuated phagocytic activity of monocytes in type 2 diabetic Goto-Kakizaki rats

The aim of this study was to determine whether phagocytic activity of leukocytes is altered in type 2 diabetes. Goto-Kakizaki (G-K) rats, a genetic model for type 2 diabetes, and Wistar rats (control) were used to analyze the immunological status of phagocytes. Direct analysis of phagocytes was performed using peripheral whole blood. Phagocytic activity of monocytes induced by Escherichia coli BioParticles was significantly lower in G-K rats than in the control rats, whereas no significant differences in phagocytic activity of granulocytes and lymphocytes were found between G-K and control rats. Monocytes of G-K rats showed significantly lower CD11b/c expression compared with that in monocytes of control rats. However, lipopolysaccharide-stimulated activation of extracellular signal-regulated kinase and nuclear factor-κB in monocytes was not significantly different between G-K and control rats. Restriction of diet in G-K rats greatly improved their hyperglycemic status, but did not restore the levels of phagocytic activity and CD11b/c expression in monocytes of G-K rats to the levels observed in control rats. The results suggest that the phagocytic activity of monocytes is attenuated in G-K rats and that this attenuation is independent of blood glucose levels and is partly explained by a decrease in CD11b/c expression in G-K rats.