Regionally different immunoreactivity for Smurf2 and pSmad2/3 in TDP-43-positive inclusions of amyotrophic lateral sclerosis.

AIMS: Smad ubiquitination regulatory factor-2 (Smurf2), an E3 ubiquitin ligase, can interact with Smad proteins and promote their ubiquitin-dependent degradation, thereby controlling the cellular levels of these signalling mediators. We previously reported that phosphorylated Smad2/3 (pSmad2/3) was sequestered in transactive response DNA-binding protein-43 (TDP-43) inclusions in the spinal cord of patients with amyotrophic lateral sclerosis (ALS). Recent biochemical and immunohistochemical studies on spinal cord and brain of ALS patients demonstrated that the composition of the TDP-43 inclusions is regionally distinct, suggesting different underlying pathogenic processes. We aimed to elucidate regional differences in pathomechanisms and composition of TDP-43 inclusions in relation to pSmad2/3 and Smurf2. METHODS: The spinal cord and brain tissues of 13 sporadic ALS (SALS) patients were investigated using immunohistochemical analysis. RESULTS: TDP-43-positive inclusions in lower motor neurones of SALS patients were immunopositive for Smurf2 and pSmad2/3. Multiple immunofluorescence staining for Smurf2, pSmad2/3, TDP-43 and ubiquitin revealed co-localization of these four proteins within the inclusions in lower motor neurones of SALS patients. Furthermore, the loss of nuclear pSmad2/3 immunoreactivity was observed in cells bearing TDP-43 inclusions. In contrast, TDP-43-positive inclusions in the extramotor neurones in the brain of SALS patients were noticeably negative for Smurf2 and pSmad2/3. In addition, pSmad2/3 immunoreactivity was preserved in the nuclei of inclusion-bearing cells. CONCLUSIONS: This regional difference in the expression of Smurf2 and pSmad2/3 within TDP-43-positive inclusions might be one of the pathomechanisms underlying the loss of lower motor neurones and comparatively spared cortical neurones seen in ALS.

Levodopa challenge test and (123) I-metaiodobenzylguanidine scintigraphy for diagnosing Parkinson's disease.

OBJECTIVES: To explore the possibility of a generally applicable tool for the immediate diagnosis of Parkinson's disease (PD) in its early stage, we compared the sensitivity and specificity of an acute levodopa challenge test with that of (123) I-metaiodobenzylguanidine (MIBG) myocardial scintigraphy. MATERIALS AND METHODS: A consecutive series of 45 patients with extrapyramidal symptoms were recruited to the acute levodopa challenge and evaluated for improvement by use of the Unified Parkinson's Disease Rating Scale motor scores. Of these patients, 32 of them were also examined by MIBG scintigraphy. The patients were followed up for at least 24 months, and 22 patients were diagnosed as having clinically definite PD. RESULTS: The sensitivity and specificity of the acute levodopa challenge test to predict clinical diagnosis of PD were 81.8% and 81.8%, respectively, which were better than those obtained by MIBG scintigraphy (62.5% and 62.5%). In both early- and middle-stages of PD, the test gave better sensitivity than MIBG scintigraphy. CONCLUSIONS: Considering that the well-established and frequently referred clinical diagnostic criteria require longitudinal observation for at least 24 months, the acute levodopa challenge test can be used as an immediate diagnostic tool for PD with sensitivity and specificity comparable to those of MIBG.

Factors which influence development of macrophage-layer and spleen colonies in mice.

Irradiated mice infused with bone marrow cells developed colonies both in the spleen and on the macrophage layer of the cellulose acetate membrane which had been inserted into their peritoneal cavity. Factors influencing the ratio of spleen to macrophage-layer colonies were examined. The ratio of the colonies depended on the condition of the recipients rather than the condition of the injected cells provided there was no marked histoincompatibility between donors and recipients.

Two necropsy cases of hypertrophic cardiomyopathy in Holstein cattle.

Two cases of hypertrophic cardiomyopathy in Holstein dairy cows are presented. At necropsy, the hearts revealed proportionate hypertrophy of the entire ventricles. The cut surface showed relatively large areas of myocardial scarring scattered throughout the ventricular walls including the septum. Microscopic examination revealed marked disorganization of cardiac muscle cells, intramural coronary arteries with thickened walls and narrowed lumina, and pronounced myocardial fibrosis. These features resemble those of hypertrophic cardiomyopathy in humans, suggesting the presence of a similar primary myocardial disease in cattle.

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.

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 modification of manganese porphyrins with biomolecules for new functional antioxidants.

Superoxide dismutase (SOD), which catalyzes the reduction of O2*- to H2O2, is the key enzyme for the protection of oxidative stress. Here we have chemically modified manganese (Mn) porphyrins with biomolecules for new functional antioxidants. The Mn-porphyrins were conjugated with the following biochemical functional molecules: (1) catalase, to catalyze reduction of H2O2 to H2O. The resulting conjugate showed dual functions of SOD and catalase; (2) a carbohydrate, to facilitate receptor binding and, hence, active targeting. The resulting conjugate showed both SOD activity and carbohydrate recognition. These results suggest that the antioxidants promise the application to biomedical fields.

Colony-forming ability of bone marrow cells of W anaemic mice on macrophage layer formed in peritoneal cavity of mice.

The colony-forming ability of haematopoietic cells of W anaemic mice was examined on the macrophage layer formed in the peritoneal cavity of mice. Bone marrow cells of W anaemic mice formed a considerable number of colonies on the macrophage layer, notwithstanding they did not form any colonies in the spleen of the same recipients. As the colony-forming ability of the bone marrow cells was not reduced by the incubation with 3-H-thymidine, most of the cells which formed colonies on the macrophage layer seemed to stay in G0 state. The interrelationship between the spleen colony-forming cells, the macrophage-layer colony-forming cells, and in vitro colony-forming cells was discussed.

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.

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.