Electrotransfer of human IL-1Ra into skeletal muscles reduces the incidence of murine collagen-induced arthritis

BACKGROUND: It has previously been demonstrated that high levels of gene expression in skeletal muscles can be achieved after direct in vivo electrotransfer of naked plasmid DNA. The purpose of this study is to examine the potential of in vivo electroporation of plasmid DNA encoding human IL-1Ra for the prevention of murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were injected in gastrocnemius muscles with plasmid DNA followed by in vivo electroporation. To uncover the optimum conditions of gene transfer, various electric field strengths and different amounts of plasmid DNA were applied. Calf muscles around the injected areas were investigated with histological methods for damage to muscle tissue. The levels of human IL-1Ra expression in the injected area and also in the serum were determined with ELISA for human IL-1Ra. Based on these data, the effects of electrotransfer of plasmid DNA were tested using the murine CIA model. DBA/1 mice were immunized with bovine collagen type II at the base of the tail. On day 21, mice were given a booster injection with the same antigen. Mice were divided into two groups on day 26. One group of mice received plasmid containing the IL-1Ra cDNA sequence, while control mice were given plasmid lacking the IL-1Ra coding sequence. The incidence of arthritis was evaluated by macroscopic analysis, histological analysis, and the levels of inflammatory cytokines. RESULTS: IL-1Ra expression increased as a function of the electrical field strength and the amount of DNA. 200 V/cm (eight pulses; 20 ms per pulse; 1 Hz) and 15 microg of plasmid DNA per mouse were found to be optimum for gene transfer. After in vivo electroporation, gene expression in both muscle and serum increased gradually, reaching a peak value on day 10. Significant levels of human IL-1Ra expression were maintained for 20 days. Macroscopic analysis showed that the onset of CIA was significantly inhibited by direct electrotransfer of plasmid DNA encoding human IL-1Ra. Histological analysis of knee joints showed that the incidence of arthritis in knee joints was also prevented. The levels of mouse IL-1beta and IL-12 in paws were significantly lower in the group treated with IL-1Ra than those in the control group. CONCLUSIONS: These results demonstrate that direct electrotransfer of plasmid containing the human IL-1Ra cDNA sequence to skeletal muscle can reduce the incidence of CIA in mice.     

PowerCore: a program applying the advanced M strategy with a heuristic search for establishing core sets

MOTIVATION: Core sets are necessary to ensure that access to useful alleles or characteristics retained in genebanks is guaranteed. We have successfully developed a computational tool named 'PowerCore' that aims to support the development of core sets by reducing the redundancy of useful alleles and thus enhancing their richness. RESULTS: The program, using a new approach completely different from any other previous methodologies, selects entries of core sets by the advanced M (maximization) strategy implemented through a modified heuristic algorithm. The developed core set has been validated to retain all characteristics for qualitative traits and all classes for quantitative ones. PowerCore effectively selected the accessions with higher diversity representing the entire coverage of variables and gave a 100% reproducible list of entries whenever repeated. AVAILABILITY: PowerCore software uses the .NET Framework Version 1.1 environment which is freely available for the MS Windows platform. The files can be downloaded from http://genebank.rda.go.kr/powercore/. The distribution of the package includes executable programs, sample data and a user manual.     

New Lignans from the Flower of Forsythia koreana and Their Suppression Effect on VCAM‐1 Expression in MOVAS Cells

Six lignans including two new lignans were obtained as the principal components of the Forsythia koreana flowers via silica gel (SiO₂), octadecyl SiO₂ (ODS) as well as Sephadex LH‐20 column chromatography. In addition to two new lignans, named koreanaside A ((7R,8S,7′R,8′S)‐7,7′‐diepoxy‐5′‐hydroxy‐3,3′‐dimethoxylignan 4‐O‐β‐d ‐glucopyranoside) and koreanaside B ((7R,8S,7′S,8′R)‐7,9′‐epoxy‐9,5′,7′‐trihydroxy‐3,3′‐dimethoxylignan 4‐O‐β‐d ‐glucopyranoside), four known lignans were identified to be (+)‐phylligenin, (?)‐epipinoresinol, pinoresinol, and tinosposide A. The structures and absolute configurations of koreanasides A and B were established by means of analysis of spectroscopic data (NMR, IR, FAB‐MS, and CD), whereas the structures of known lignans were identified by comparison their NMR and MS values with those in the reported literature. Their chemical structures including configuration were established by means of analysis of spectroscopic data (NMR, IR, FAB‐MS, and CD) but also comparison of their NMR and MS values with those in the reported literature. This is the first article for isolation of six lignans of F. koreana flowers. Koreanasides A and B showed high radical scavenging activity with oxygen radical absorbance capacity (ORAC) values of 0.97 ± 0.01 and 1.02 ± 0.01, respectively. Koreanaside A also prohibited expressing VCAM‐1 in MOVAS cells with 80.5% at 25 mg/mL.     

Assessment of genetic diversity and population structure in mungbean

This study was carried out to assess the genetic diversity and to analyze the population genetic structure for a total of 692 mungbean accessions preserved at National Agrobiodiversity Center (NAC) of the Rural Development Administration (RDA), Korea. Mungbean accessions were collected from 27 countries in nine different geographic regions, and were genotyped using 15 microsatellite markers, which were developed in our previous study. A total of 66 alleles were detected among 692 accessions at all the loci with an average of 4.4 alleles per locus. All the microsatellite loci were found to be polymorphic. The expected heterozygosity (H _E ) and polymorphism information content (PIC) ranged from 0.081 to 0.588 (mean = 0.345) and from 0.080 to 0.544 (mean = 0.295), respectively. Of the 66 alleles, 17 (25.8%) were common (frequency range between 0.05 and 0.5), 15 (22.7%) were abundant (frequency range > 0.5), and 34 (51.5%) were rare (frequency range < 0.05). Locus GB-VR-7 provided the highest number of rare alleles(eight), followed by GB-VR-91(six) and GB-VR-113(four). Country-wide comparative study on genetic diversity showed that accessions from the USA possessed the highest genetic diversity (PIC) followed by Nepal, Iran, and Afghanistan. And region-wide showed that accessions from Europe possessed the highest average genetic diversity, followed by accessions from the USA, South Asia, West Asia, and Oceania. Twenty-seven countries were grouped into seven clades by phylogenetic relationship analysis, but clustering pattern did not strictly follow their geographical origin because of extensive germplasm exchange between/among countries and regions. As a result of a model-based analysis (STRUCTURE) of microsatellite data, two distinct genetic groups were identified which shared more than 75% membership with one of the two genetic groups. However the genetic group pattern did not reflect their geographical origin. The Duncan’s Multiple Range Test among these two genetic groups and an admixed group, with a mean of 16 phenotypic traits, showed significant difference in 12 quantitative and qualitative traits on the basis of ANOVA. These 15 newly developed SSR markers proved to be useful as DNA markers to detect genetic variation in mungbean germplasm for reasonable management and crossbreeding purposes.     

Intrasplenic electro-transfer of IL-4 encoding plasmid DNA efficiently inhibits rat experimental allergic encephalomyelitis

Most of the previous studies in which cytokine DNA plasmids were delivered by systemic administration exhibited only marginal therapeutic effects, if any, in the EAE model. One strategy to overcome this limitation would be to determine the optimal delivery route leading to significant beneficial effects both in early (prophylactic) and late (therapeutic) treatments. To address this issue, we directly compared the effects of intrasplenic (i.s.) and intramuscular (i.m.) electro-transfer of interleukin-4 (IL-4) DNA in the rat experimental allergic encephalomyelitis (EAE) model. In the preventive experiment, rats received i.m. (25 or 150 microg) or i.s. (25 microg) administration of IL-4 DNA followed by in vivo electroporation the day before MBP immunization. In the late treatment experiment, rats were treated with i.m. (150 microg) or i.s. (25 microg) administration of IL-4 DNA with electroporation 10 days after MBP immunization. As a control the same amount of vector DNA was used. Macroscopic analysis indicated that the onset of moderate to severe EAE in rats treated with i.s. transfer of 25 microg of IL-4 DNA was prevented on a significant level compared with i.m. 25 microg of the IL-4 DNA transfer group or the control group in the preventive experiments. More importantly, i.s. transfer of 25 microg of IL-4 DNA considerably suppressed the severity of EAE in late treatment experiments while i.m. transfer of 150 microg of IL-4 DNA had little effect. The MBP-specific expression of IFN-gamma from stimulated splenocytes was considerably decreased by the i.s. IL-4 DNA transfer group both in the preventive and therapeutic experiments while i.m. transfer had this effect only in the preventive protocol. Histological analysis showed that spinal cord inflammation was considerably reduced in the i.s. IL-4 DNA transfer group. These data provide the first demonstration that i.s. electro-transfer of IL-4 DNA is more effective both in the prevention and modulation of EAE than i.m. transfer and that i.s. electro-gene transfer may present a new approach to cytokine therapy in autoimmune diseases.     

Ca2+-Mediated Activation of c-Jun N-Terminal Kinase and Nuclear Factor κB by NMDA in Cortical Cell Cultures

Abstract: We examined the possibility that c-Jun N-terminal kinase (JNK) and nuclear factor κB (NF-κB) might be involved in intracellular signaling cascades that mediate NMDA-initiated neuronal events. Exposure of cortical neurons to 100 µ M NMDA induced activation of JNK within 1 min. Activity of JNK was further increased over the next 5 min and then declined by 30 min. Similarly, ionomycin, a selective Ca²⁺ ionophore, induced activation of JNK. The NMDA-induced activation of JNK was abrogated in the absence of extracellular Ca²⁺, suggesting that Ca²⁺ entry is necessary and sufficient for the JNK activation. Immunohistochemistry with anti-NF-κB antibody demonstrated nuclear translocation of NF-κB within 5 min following NMDA treatment. NMDA treatment also enhanced the DNA binding activity of nuclear NF-κB in a Ca²⁺-dependent manner. Treatment with 3 m M aspirin blocked the NMDA-induced activation of JNK and NF-κB. Neuronal death following a brief exposure to 100 µ M NMDA was Ca²⁺ dependent and attenuated by addition of aspirin or sodium salicylate. The present study suggests that Ca²⁺ influx is required for NMDA-induced activation of JNK and NF-κB as well as NMDA neurotoxicity. This study also implies that aspirin may exert its neuroprotective action against NMDA through blocking the NMDA-induced activation of NF-κB and JNK.     

Seasonal biochemical plasticity of a flight muscle in a bat, Murina leucogaster

Cellular and biochemical responses of the pectoral muscle to variation in seasonal activity were studied in the bat, Murina leucogaster ognevi. We collected bats in mid-hibernation (February), end-hibernation (April), and mid-summer (August) to track major activity periods in their annual cycle. Our findings indicated that myofiber cross-sectional area decreased to 68% between mid- and end-hibernation, but returned to the winter level in mid-summer. Total soluble protein and total RNA concentrations were not altered over these sampling periods. Oxidative potential gauged by citrate synthase activity increased 1.47-fold from mid- to end-hibernation and then remained at the similar level in mid-summer. Glycolytic potential gauged by lactate dehydrogenase activity changed little between mid- and end-hibernation but increased 1.42-fold in summer, compared with the winter level. Thus, the myofibers underwent disuse atrophy during hibernation, while enzymatic catalytic function recovered towards the level of mid-summer.     

Induction and attenuation of neuronal apoptosis by proteasome inhibitors in murine cortical cell cultures

Evidence has accumulated showing that pharmacological inhibition of proteasome activity can both induce and prevent neuronal apoptosis. We tested the hypothesis that these paradoxical effects of proteasome inhibitors depend on the degree of reduced proteasome activity and investigated underlying mechanisms. Murine cortical cell cultures exposed to 0.1 microM MG132 underwent widespread neuronal apoptosis and showed partial inhibition of proteasome activity down to 30-50%. Interestingly, administration of 1-10 microM MG132 almost completely blocked proteasome activity but resulted in reduced neuronal apoptosis. Similar results were produced in cortical cultures exposed to other proteasome inhibitors, proteasome inhibitor I and lactacystin. Administration of 0.1 microM MG132 led to activation of a mitochondria-dependent apoptotic signaling cascade involving cytochrome c, caspase-9, caspase-3 and degradation of tau protein; such activation was markedly reduced with 10 microM MG132. High doses of MG132 prevented the degradation of inhibitor of apoptosis proteins (IAPs) cIAP and X chromosome-linked IAP, suggesting that complete blockade of proteasome activity interferes with progression of apoptosis. In support of this, addition of high doses of proteasome inhibitors attenuated apoptosis of cortical neurons deprived of serum. Taken together, the present results indicate that inhibition of proteasome activity can induce or prevent neuronal cell apoptosis through regulation of mitochondria-mediated apoptotic pathways and IAPs.     

Analysis of mitochondrial free radical generation in animal models of neuronal disease

Mitochondria, the power plant of all eukaryotic cells, produce cellular energy in the form of ATP via electron transport and oxidative phosphorylation. However, the mitochondria leak electrons that can act as major sources of oxidative stress, and their dysfunction, have been proposed as causative events underlying neurodegeneration in stroke and neurodegenerative diseases. We examined whether MitoTracker Red CM-H(2)XRos, a rosamine derivative used to detect mitochondrial free radicals in vitro, would be applied to analyze the mitochondrial free radicals in various models of neurological diseases in vivo. The injections of MitoTracker Red CM-H(2)XRos revealed generation of mitochondrial free radicals primarily in vulnerable neurons following focal cerebral ischemia as well as administration of Fe(2+) or 3-nitropropionic acid. MitoTracker Red CM-H(2)XRos was retained after fixation, compatible with immunocytochemistry or nuclear staining, and can be applied to study roles of mitochondrial free radicals in the process of neurodegeneration in vivo.