Spinal segmental levels innervated by different types of vestibulo-spinal tract neurones in rabbit.
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Animals↗
Biomedical subjects
T Akaike
Publications and source records attributed to T Akaike.
Role of free radicals in viral pathogenesis and mutation.
Oxygen radicals and nitric oxide (NO) are generated in excess in a diverse array of microbial infections. Emerging concepts in free radical biology are now shedding light on the pathogenesis of various diseases. Free-radical induced pathogenicity in virus infections is of great importance, because evidence suggests that NO and oxygen radicals such as superoxide are key molecules in the pathogenesis of various infectious diseases. Although oxygen radicals and NO have an antimicrobial effect on bacteria and protozoa, they have opposing effects in virus infections such as influenza virus pneumonia and several other neurotropic virus infections. A high output of NO from inducible NO synthase, occurring in a variety of virus infections, produces highly reactive nitrogen oxide species, such as peroxynitrite, via interaction with oxygen radicals and reactive oxygen intermediates. The production of these various reactive species confers the diverse biological functions of NO. The reactive nitrogen species cause oxidative tissue injury and mutagenesis through oxidation and nitration of various biomolecules. The unique biological properties of free radicals are further illustrated by recent evidence showing accelerated viral mutation by NO-induced oxidative stress. NO appears to affect a host's immune response, with immunopathological consequences. For example, NO is reported to suppress type 1 helper T cell-dependent immune responses during infections, leading to type 2 helper T cell-biased immunological host responses. NO-induced immunosuppression may thus contribute to the pathogenesis of virus infections and help expansion of quasispecies population of viral pathogens. This review describes the pathophysiological roles of free radicals in the pathogenesis of viral disease and in viral mutation as related to both nonspecific inflammatory responses and immunological host reactions modulated by NO.
Animals↗
Xenoimplantation using a diffusion chamber with a B-cell line (MIN6) as a bioartificial endocrine pancreas (Bio-AEP).
This experiment was designed to investigate whether MIN6-cells, which are derived from transgenic mouse insulinoma cells, could be a useful tool for transplantation. An implantable diffusion chamber for a bioartificial endocrine pancreas (Bio-AEP) was constructed by placing a pancreatic B-cell line (MIN6) in a mixed matrix in the center of a ring holder sandwiched between two nuclepore membranes (pore size 0.2 microns), which were held in place by a silicon seal. Nine streptozotocin (STZ)-induced diabetic rats were each implanted with the diffusion chamber containing mouse insulinoma cells (xenograft-implantation) as a Bio-AEP, but without any immunosuppressant. In three of the STZ-diabetic rats with a Bio-AEP, a return to normoglycemia was observed up to 20 week after implantation (good control). Four of the nine STZ-diabetic rats, which had received diffusion chambers with MIN6, also showed a return to normoglycemia for up to 10 wk (fair control). In two of the rats, blood glucose levels showed poor control. The results indicate that MIN6 cells should be useful for the implantation of xenographic cells in diabetic animals.
Animals↗
Targeted gene transfer to sinusoidal endothelial cells and expression in vivo.
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Animals↗
Pegylated zinc protoporphyrin: a water-soluble heme oxygenase inhibitor with tumor-targeting capacity.
Heme oxygenase (HO) is a key enzyme in heme metabolism; it oxidatively degrades heme to biliverdin, accompanied by formation of free iron and carbon monoxide. Biliverdin is subsequently reduced by cytosolic biliverdin reductase to form bilirubin, a potent antioxidant. We recently found that tumor cells utilize HO to protect themselves from oxidative stress by producing the antioxidant bilirubin. This result suggested an important potential therapeutic strategy: suppression of bilirubin production with the use of HO inhibitors; hence, cancer cells become vulnerable to oxidative stress induced by anticancer drugs or leukocytes of the host. This concept was validated by using the intraarterial administration of an HO inhibitor, zinc protoporphyrin, in nonphysiological solution. In the present study, zinc protoporphyrin (ZnPP) was conjugated with poly(ethylene glycol) (PEG) with molecular weight of 5000, to make ZnPP, a water-soluble compound (PEG-ZnPP), and to improve its tumor-targeting efficiency. PEG was conjugated to ZnPP through newly introduced amino groups, where ethylenediamine residues were added at C6 and C7 of protoporphyrin. The divalent zinc cation was chelated into the protoporphyrin ring to obtain PEG-ZnPP. PEG-ZnPP did become highly water-soluble, and it formed multimolecular associations with molecules larger than 70 kDa in aqueous media. PEG-ZnPP inhibited splenic microsomal HO activity in vitro in a competitive manner in the presence of hemin, with an apparent inhibitory constant of 0.12 microM. Most important, PEG-ZnPP injected intravenously significantly suppressed intratumor HO activity in a murine solid tumor model, which suggests that tumor-targeted inhibition of HO is possible with the use of PEG-ZnPP.
Animals↗
Comb-type polycations effectively stabilize DNA triplex.
DNA triplex formation has been studied as a potential strategy for regulation of gene expression. The triplex is, however, unstable under physiological conditions, so that an effective stabilizer for the triplex formation is needed. Here is shown a novel strategy to stabilize the triplex based on the molecular design of a comb-type polycation. Linear polycations, such as poly(L-lysine) and poly(L-arginine), thermally stabilize DNA duplexes (and triplexes). The complexes between DNA and the polycation are irreversible and are liable to precipitate out of aqueous media. The irreversibility and phase separating properties of the complex impede association of single-stranded (ss) DNAs in the complex to form duplexes and triplexes. A comb-type polycation consisting of a poly(L-lysine) backbone and grafted chains of hydrophilic polymers was prepared. The comb-type copolymers increased solubility of their complex with DNA and suppressed conformational changes of DNA. Thermal melting curve analyses revealed that the comb-type copolymer markedly stabilized DNA triplexes and did not disturb ssDNAs in forming duplexes and triplexes. Reversible and one-step melting/reassociation transitions of poly(dA).2poly(dT) triplex were shown in the pressure of the copolymers. The stabilizing effect of the copolymer was larger than that of spermine, a polyamine considered effective in stabilizing triplexes. These results indicated that molecular design of polycations with a comb-type structure is a novel strategy to create efficient triplex stabilizers. Such comb-type copolymer consisting of various types of polycation backbones and hydrophilic graft chains may have many applications in which specific and precise interactions of polynucleotides are involved.
Animals↗
Design of comb-type polyamine copolymers for a novel pH-sensitive DNA carrier.
The comb-type polycation consisting of a poly[2-(diethylamino)ethyl methacrylate] (PDEAEMA) backbone and poly(L-lysine) (PLL) side chains has been prepared as a novel pH-sensitive DNA carrier. The comb-type copolymer PDEAEMA-graft-PLL was prepared by using the macromonomer method, in which a poly(N epsilon-carbobenzoxy-L-lysine) macromonomer was radically copolymerized with DEAEMA. The comb-type copolymer exhibited a two-step proton dissociation and a dual ionic character owing to the two cationic segments in the copolymer, as determined by acid-base titration. In addition, the comb-type copolymer caused no significant turbidity even at pH 10, whereas PDEAEMA homopolymer suddenly precipitated out of the aqueous medium above pH 7.5 owing to the deprotonation of amino groups. Furthermore, a 1H NMR study proved that protonated PLL segments solubilized the comb-type copolymer with a hydrophobic PDEAEMA core at higher pH. Finally, the pH-dependent behavior of the DNA complex with the comb-type copolymer was evaluated. The discontinuous turbidity change of the DNA/PDEAEMA-graft-PLL mixture at pH 7.5 suggested that the solubility of the complex varied in response to pH. By circular dichroism measurement, we also found that the comb-type copolymer was capable of varying DNA compaction pH-dependently. In conclusion, we have demonstrated that the comb-type copolymer is capable of sensing a pH signal and outputting the nonlinear change of the physicochemical properties of DNA polyelectrolyte complexes.
Chemical Phenomena↗
Nanoparticle DNA carrier with poly(L-lysine) grafted polysaccharide copolymer and poly(D,L-lactic acid).
Biodegradable nanoparticles, which contain the sites for both polynucleotide adsorption and targeting ligand on their surfaces, were prepared as a novel carrier for genetic materials. The nanoparticles were obtained from poly(D,L-lactic acid) and poly(L-lysine)-graft-polysaccharide copolymers by using either a solvent evaporation method or a diafiltration method. The size of the particles prepared by the diafiltration method was controlled by varying the initial concentration of the graft copolymer. Nanoparticles as small as 60 nm in diameter were successfully obtained from the graft copolymers with high polysaccharide contents but not from the poly(L-lysine) homopolymer. Polysaccharide moieties on the surface of the nanoparticles were found to interact specifically with a particular lectin as verified by the aggregation assay. The polynucleotide adsorption capacity of the nanoparticles was increased with increasing polysaccharide contents in the graft copolymers, suggesting that the adsorption conformation of poly(L-lysine) moiety in the graft copolymer on the nanoparticle surface is different from that in poly(L-lysine) homopolymer. Moreover, the nanoparticles from the graft copolymer exhibited resistance against self-aggregation and nonspecific adsorption of serum proteins, presumably due to the polymer brush effect and/or exclusion effect from the polysaccharide graft chains. These results suggest that the nanoparticles prepared from poly(L-lysine)-graft-polysaccharide copolymer and poly(D,L-lactic acid) can serve as a good DNA carrier in vivo.
Adsorption↗
Characterization of interpolyelectrolyte complexes between double-stranded DNA and polylysine comb-type copolymers having hydrophilic side chains.
The polyionic interaction between DNA and polycations grafted with hydrophilic dextran side chains was evaluated. The comb-type copolymers, poly(L-lysine)-graft-dextran, were successfully prepared by employing a reductive amination reaction between epsilon-amino groups of poly(L-lysine) (PLL) and the reductive ends of dextran (Dex). A coupling efficacy on the order of 70% was obtained regardless of intrinsic philicities of the solvents used, either aqueous buffer or DMSO. The resulting graft copolymers, which varied in the degree of grafting and the length of hydrophilic side chains, formed a soluble complex with DNA. They also affected the melting behavior of double-stranded DNA (dsDNA) in different ways. Copolymers having a high degree of grafting thermally stabilized dsDNA without affecting its reversible transition between single-stranded and double-stranded forms. However, copolymers with a low degree of grafting or with a high degree of grafting of short dextran chains impeded the reversibility of this transition. Furthermore, highly grafted copolymers also accelerated the hybridization of DNA strands in a low-ionic strength medium. It is of particular note that these copolymers scarcely altered circular dichroismic signals of dsDNA even when the copolymers were added in excess. This suggested that the copolymer interacted with dsDNA without affecting its native structure or physicochemical properties. Finally, the copolymer even formed a stable complex with a short oligonucleotide (20 bases). We, therefore, concluded that, by regulating the degree of grafting and the molecular weight of grafted side chains, it would be possible to design novel different graft copolymers capable of acting as carriers of functional genes to target cells or tissue.
Animals↗
Synthesis of novel polyampholyte comb-type copolymers consisting of a poly(L-lysine) backbone and hyaluronic acid side chains for a DNA carrier.
The polyampholyte comb-type copolymers consisting of a poly(L-lysine) (PLL) main chain, a DNA binding site, and hyaluronic acid (HA) side chains, cell-specific ligands, have been prepared as the DNA carrier targeting sinusoidal endothelial cells of liver. The reducing end of HA and epsilon-amino groups of PLL were covalently coupled by reductive amination to obtain the resulting comb-type copolymers (PLL-graft-HA). The chain length of HA was controlled by the enzymatic hydrolysis of high-molecular weight HA. Since HA formed polyion complexes with PLL, the coupling reaction was carried out with high-ionic strength media to suppress polyion complex formation. The reaction proceeded in a homogeneous system, leading to a high efficiency of coupling (>70%) of HA onto the PLL backbone. By using the enzymatic hydrolysis of HA and the reductive amination reaction between HA and PLL with high-ionic strength media, it is possible to prepare the various comb-type copolymers with a defined density and a defined length of HA side chains. Furthermore, we also find that these polyampholyte comb-type copolymers vary their assembling structure in water in response to two kinds of environmental factors, i.e., ionic strength and pH. Finally, a 1H NMR study reveals that the PLL backbone efficiently interacts with DNA molecules despite the presence of HA side chains having negative charges.
Carbohydrate Sequence↗
Comb-type prepolymers consisting of a polyacrylamide backbone and poly(L-lysine) graft chains for multivalent ligands.
The comb-type copolymers consisting of a polyacrylamide (PAAm) backbone and poly(L-lysine) (PLL) graft chains have been prepared as the "prepolymer" for designing multivalent ligands. To regulate the length and density of the clusters of primary amino groups, the Nalpha-carboxyanhydride of Nepsilon-carbobenzoxy (CBZ)-L-lysine was first polymerized using p-vinylbenzylamine as an initiator. The resulting poly(CBZ-L-lysine) macromonomer was then radically copolymerized with AAm, followed by the deprotection of amino groups. For the model study, the reactive clusters of primary amino groups were completely converted into anion clusters by the reaction with succinic anhydride. The model multivalent ligands having the biotin label on the PAAm backbone were prepared by the terpolymerization of the macromonomer, AAm, and the biotin derivative having a vinyl group. The enzyme-linked immunosorbent assay showed that the biotin with no spacer on the PAAm backbone was recognized by the avidin-peroxidase conjugate specifically. Therefore, the highly sensitive detection of the interaction between cells and various model multivalent ligands was possible. The selective labeling onto the PAAm backbone revealed that the converted anion clusters of graft chains interacted exclusively with the cell and that the backbone was inert to the interaction with the cell. These results indicate that the various PAAm-graft-PLL comb-type copolymers with the defined length and density of the PLL-grafts are the potential prepolymers to investigate and to optimize the affinity of the multivalent ligands for receptors.
Acrylamide↗
Mechanism of intermolecular purine-purine-pyrimidine triple helix stabilization by comb-type polylysine graft copolymer at physiologic potassium concentration.
We previously reported a novel strategy to stabilize purine motif triplex DNA within a mammalian gene promoter at physiologically relevant pH, temperature, and potassium (K(+)) concentrations by a comb-type poly(L-lysine)-graft-dextran copolymer [Ferdous et al., (1998) Nucleic Acids Res. 26, 3949-3954]. Here we describe the major contribution(s) of the copolymer to stabilize the purine motif triplex DNA at physiological K(+) concentrations. Self-aggregation through guanine-quartet formation of guanine-rich (G-rich) triplex-forming oligonucleotides (TFOs) has long been proposed for K(+)-mediated inhibition of the purine motif triplex formation. However, this was not the case for the severe inhibitory effect of K(+) observed under our reaction conditions. Rather significant decrease in rate of triplex formation involving a G-rich TFO was a major factor to confer K(+) inhibition. Interestingly, in the presence of the copolymer the rate of triplex formation was tremendously increased and K(+)-induced dissociation of preformed triplexes was not observed. Moreover, the triplex-promoting/stabilizing efficiency of the copolymer was amazingly higher than that of physiological concentrations of spermine. An absolute increase in binding constant of the TFO to the target duplex could therefore be the predominant mechanistic source for the copolymer-mediated triplex stabilization under physiological conditions in vitro.
Base Sequence↗
Relative effects of graft copolymer and polyamines on triplex stabilization under physiological conditions.
Triplex-stabilizing effect of a graft copolymer under physiologically relevant conditions has been evaluated and compared with other polyamines. Here we show that the graft copolymer significantly stabilizes triplex DNAs with amazingly higher efficacy than that of physiological concentrations of spermine and spermidine.
Base Sequence↗
Comb-type copolymers for controlled DNA delivery.
Various comb-type copolymer containing a polycation as a main chain was design to construct delivery systems of DNAs. The comb-type copolymers having cell-specific polysaccharides were proved to be useful to deliver DNA to the target cells in vivo. Of interest, the copolymers with abundant side chains of hydrophilic polymers are capable of stabilizing DNA triplex. Further, injectable nanoparticles for controlled releases of DNAs were fabricated from the copolymer and a biodegradable polymer.
Biocompatible Materials↗
The protective effect of pyrroloquinoline quinone and its derivatives against carbon tetrachloride-induced liver injury of rats.
Pyrroloquinoline quinone (PQQ) and its derivative, oxazo pyrroloquinoline (OPQ-G), protected rats from experimental liver injury induced by carbon tetrachloride (CCl4) in vivo. This effect was observed after an intraperitoneal injection of 5 mg/kg PQQ or OPQ-G, which was given twice, 10 min and 1 h before CCl4 administration. Pyrroloquinoline quinone protected primary cultured rat hepatocytes from CCl4 toxicity in vitro. This protection was most effective at a concentration of 3 mumol/L PQQ. Pyrroloquinoline quinone derivatives (oxazo pyrroloquinoline, methyl-thioethyl oxazo pyrroloquinoline and PQQ-allylester) also protected the hepatocytes from CCl4 toxicity. Pyrroloquinoline quinone and its derivatives inhibited the lucigenin-enhanced chemiluminescence from isolated hepatocytes initiated by CCl4. These results suggest that eliminating free radicals is one of the protective mechanisms of PQQ and its derivatives against CCl4-induced liver injury.
Acridines↗
Development of a method for embedded-culture of pig pancreatic islet-like cell clusters in agarose containing maltose-carrying polystyrene (HEVM) and nicotinamide.
A method for embedded-culture of islet-like cell clusters (ICCs) from neonatal pig pancreas is described. The procedure is based on a sequential treatment of the pancreas with a proteolytic enzyme and ethylenediaminetetraacetate (EDTA). During the exposure to EDTA-Dispase, small pieces of pancreas were gradually digested, with gentle stirring. Under- and over-digested pancreatic cell clusters were separated from the pancreatic fragments by filtration, and formed whole islet-like cell clusters in RPMI 1640 containing 11.0 mM D-glucose and 10% fetal bovine serum (FBS). The ICCs were embedded in agarose with or without a gel containing a random copolymer hydroxethylmethacrylate-vinylbenzyl maltonamide (HEVM) of hydroxethylmethacrylate-(HEMA)-vinylbenzyl maltonamide (VMA) and nicotinamide. They remained morphologically intact and a physiological response to acute stimulation with glucose was obtained when they were embedded in the agarose containing HEVM and nicotinamide. These findings suggest that ICCs in agarose containing HEVM and nicotinamide could be a useful tool for morphological, biochemical and molecular biological studies, and also as a potential source of material for transplantation.
Animals↗
Preparation of adult pig pancreatic cells: comparative study of methods with or without proteolytic enzymes.
Methods for the preparation of adult pig pancreatic cells using enzymatic- or auto-digestion are described, and a comparison of these methods was performed. A greater number of cells staining red with dithizone with the highest viability and insulin secretory response to glucose stimulation were obtained with dispersed pancreatic cells prepared by auto-digestion of the pancreas. On the other hand, many damaged cells were observed when the pancreas was exposed to collagenase or EDTA-Dispase and only a few cells stained red with dithizone. Insulin secretion in response to glucose stimulation was observed for up to 6 h in cells prepared using the auto-digestion procedure and an approximately 2- to 3-fold increase in insulin secretion was seen in response to glucose. Cells obtained by the collagenase or EDTA-Dispase exposure did not show a secretory response to either low (5.5 mmol/l) or high (16.7 mmol/l) glucose concentrations. This report shows that it is possible to prepare pancreatic endocrine cells from the adult pig pancreas which represents an inexpensive way to obtain cells for pancreatic cell transplantation or studies of their biochemical or physiological properties.
Animals↗
Embedded-culture of pancreatic beta-cells derived from transgenic mouse insulinoma as a potential source for xenotransplantation using a diffusion chamber.
Embedded-culture of pancreatic beta-cells derived from a transgenic mouse insulinoma (MIN6 cells) was studied in vitro and in vivo. The MIN6 cells were enmeshed in an agarose-PVMA-collagen matrix for long-term maintenance. The cells formed islet-like cell clusters (ICCs) in the mixed matrix. When 10 mmol/L nicotinamide was added to these cultures the cells secreted insulin in response to various concentrations of glucose, whereas the untreated control cells were unresponsive. Both control and nicotinamide-treated MIN6 cells exhibited normal beta-cell function for up to 35 days in the mixed matrix, and the cells were much better preserved with nicotinamide than without it. MIN6 cells were suspended in the mixed matrix with nicotinamide and transferred into diffusion chambers to create a bio-artificial endocrine pancreas (Bio-AEP). In streptozotocine-induced diabetic rats with implanted Bio-AEP but without any immunosuppressants, a return to normoglycaemia was observed for up to 12 wk or more after transplantation. Our results indicate that nicotinamide-treated MIN6 cells embedded in a mixed matrix should be useful for the study of xenotransplantation and the development of a bioartificial endocrine pancreas.
Animals↗