Activation of poly(ADP-ribose) polymerase in the rat hippocampus may contribute to cellular recovery following sublethal transient global ischemia

We have investigated the role of poly(ADP-ribose) polymerase (PARP) activation in rat brain in a model of sublethal transient global ischemia. Adult male rats were subjected to 15 min of ischemia with brain temperature reduced to 34 degrees C, followed by 1, 2, 4, 8, 16, 24, and 72 h of reperfusion. PARP mRNA expression was examined in the hippocampus using quantitative RT-PCR, northern blot analysis, and in situ hybridization. Protein expression was assessed using western blot analysis. PARP enzymatic activity was investigated by measuring nuclear [3H]NAD incorporation. The presence of poly(ADP-ribose) polymers was assessed immunocytochemically. Although PARP mRNA and protein expressions were not altered after ischemia, enzymatic activity was increased 4.37-fold at 1 h (p < 0.05 vs. sham) and 1.73-fold (p < 0.05 vs. sham) at 24 h of reperfusion. Immunostaining demonstrated the presence of poly(ADP-ribose) polymers in CA1 neurons. Cellular NAD+ levels were not significantly altered at any time point. Furthermore, systemic administration of 3-aminobenzamide (30 mg/kg), a PARP inhibitor, prevented the increase in PARP activity at 1 and 24 h of reperfusion, significantly decreased the number of surviving neurons in the hippocampal CA1 region 72 h after ischemia (p < 0.01 vs. sham), and increased DNA single-strand breaks assessed as DNA polymerase I-mediated biotin-dATP nick-translation (PANT)-positive cells (p < 0.01 vs. sham). Furthermore, using an in vitro DNA repair assay, 3-aminobenzamide (30 mg/kg) was shown to block DNA base excision repair activity. These data suggest that the activation of PARP, without subsequent NAD+ depletion, following mild transient ischemia may be neuroprotective in the brain.     

Induction of oxidative DNA damage in the peri-infarct region after permanent focal cerebral ischemia

To address the role of oxidative DNA damage in focal cerebral ischemia lacking reperfusion, we investigated DNA base and strand damage in a rat model of permanent middle cerebral artery occlusion (MCAO). Contents of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and apurinic/apyrimidinic abasic sites (AP sites), hallmarks of oxidative DNA damage, were quantitatively measured in nuclear DNA extracts from brains obtained 4-72 h after MCAO. DNA single- and double-strand breaks were detected on coronal brain sections using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), respectively. Levels of 8-OHdG and AP sites were markedly elevated 16-72 h following MCAO in the frontal cortex, representing the peri-infarct region, but levels did not significantly change within the ischemic core regions of the caudateputamen and parietal cortex. PANT- and TUNEL-positive cells began to be detectable 4-8 h following MCAO in the caudate-putamen and parietal cortex and reached maximal levels at 72 h. PANT- and TUNEL-positive cells were also detected 16-72 h after MCAO in the lateral frontal cortex within the infarct border, where many cells also showed colocalization of DNA single-strand breaks and DNA fragmentation. In contrast, levels of PANT-positive cells alone were transiently increased (16 h after MCAO) in the medial frontal cortex, an area distant from the infarct zone. These data suggest that within peri-infarct brain regions, oxidative injury to nuclear DNA in the form of base and strand damage may be a significant and contributory cause of secondary expansion of brain damage following permanent focal ischemia.     

Electron cryomicroscopy of bacteriorhodopsin vesicles: mechanism of vesicle formation.

We obtained vesicles from purple membrane of Halobacterium halobium at different suspension compositions (pH, electrolytes, buffers), following the procedure of Kouyama et al. (1994) (J. Mol. Biol. 236:990-994). The vesicles contained bacteriorhodopsin (bR) and halolipid, and spontaneously formed during incubation of purple membrane suspension in the presence of detergent octylthioglucoside (OTG) if the protein:OTG ratio was 2:1 by weight. The size distribution of the vesicles was precisely determined by electron cryomicroscopy and was found to be almost independent on the incubation conditions (mean radius 17.9-19 nm). The size distribution in a given sample was close to the normal one, with a standard deviation of approximately +/- 1 nm. During dialysis for removal of the detergent, the vesicles diminished their radius by 2-2.5 nm. The results allow us to conclude that the driving force for the formation of bR vesicles is the preferential incorporation of OTG molecules in the cytoplasmic side of the membrane (with possible preferential delipidation of the extracellular side), which creates spontaneous curvature of the purple membrane. From the size distribution of the vesicles, we calculated the elasticity bending constant, K(B) approximately 9 x 10(-20) J, of the vesicle wall. The results provide some insight into the possible formation mechanisms of spherical assembles in living organisms. The conditions for vesicle formation and the mechanical properties of the vesicles could also be of interest with respect to the potential technological application of the bR vesicles as light energy converters.     

Induction of Cross-Reactivity in an Endogenous Viral Peptide Non-Reactive to FBL-3 Tumor-Specific Helper T-Cell Clones

We previously reported a helper T-cell (Th) epitope (peptide i) which corresponded to the sequence ranging from positions 462 to 479 from the N-terminus of the Friend-murine leukemia virus (F-MuLV) envelope protein (env_462-479). Homologous sequences exist in both Moloney-murine leukemia (M-MuLV env_452-469) and endogenous AKV (AKV env_453-470) viruses, which differ from F-MuLV env_462-479 in 5 and 7 amino acids, respectively. However, peptide i-specific Th clones did not respond to either of the corresponding exogenous or endogenous peptides. One amino acid substitution in M-MuLV env_452-469 (Asn to Tyr at position 465: N465Y) and three amino acids in AKV env_453-470 (H460S, A466Y and Y468H) endowed both peptides with the reactivity to one of the Th clones, F5-5, almost to the same degree as peptide i. However, the other Th clones responded differently to each of the modified endogenous peptides substituted by one to three amino acids. The cells responsive to the cross-reactive peptides occupied only a minor portion, if any, of the bulk cultured lymph node cells from peptide i-immune mice, and in particular, no significant response to the modified endogenous peptides was observed in repeated experiments. The exchange of at least 3 residues was necessary for the endogenous peptide to acquire sufficient cross-reactivity to two of the three Th clones. However, it was noticeable that a single substitution of alanine by tyrosine at the dominant T-cell receptor (TCR) contact position of the peptide i_e generated a weak but significant cross-reactivity to one of the three Th clones in this study. Thus, peptides of endogenous retroviral origin that would be modified by mutational events might become ‘non-self’ and prime Th cells leading to auto-antibody production and resulting in autoimmune disease.     

Nutrient dynamics and lignocellulose degradation in decomposing Quercus serrata leaf litter

The litter mass loss, concentration and mass of some major nutrient elements, degradation of lignin and cellulose in decomposing Quercus serrata Murray leaf litter were monitored for 3 years using the litterbag method. The mobility of elements during the course of the study was in the order of: K > P > C > Mg > Ca > N. Three patterns of nutrient dynamics were observed: (i) concentration increased while mass decreased (N, Mg and Ca); (ii) concentration and nutrient mass decreased (K and C); and (iii) both concentration and mass had fluctuated (P). The C to element ratio tended to increase as the element was released, and decreased as the element was retained. Nitrogen mobility in relation to carbon was characterized by three phases: (i) initial release; (ii) accumulation and (iii) final release. The decay rate (k) calculated from 0–6 months period was overestimated for an average annual rate while those of 0–36 months fit the negative single exponential model (Adj. r² = 0.99) better than shorter periods. For lignin, the concentration had increased then decreased but tended to stabilize after 1 year while the lignin mass had continuously decreased throughout the study period. During the first 9 months, both the concentrations and mass of cellulose had fluctuated but declined thereafter. The amounts of N had initially increased but declined after 1 year; P had fluctuated while K, Ca, Mg and C had decreased throughout the study. N and C/N ratio exerted strong influence on mass loss during the first24 months but the influence of lignin emerged after 24 months.     

Embryonic Medaka Model of Microglia in the Developing CNS Allowing In Vivo Analysis of Their Spatiotemporal Recruitment in Response to Irradiation

Radiation therapy (RT) is pivotal in the treatment of many central nervous system (CNS) pathologies; however, exposure to RT in children is associated with a higher risk of secondary CNS tumors. Although recent research interest has focused on the reparative and therapeutic role of microglia, their recruitment following RT has not been elucidated, especially in the developing CNS. Here, we investigated the spatiotemporal dynamics of microglia during tissue repair in the irradiated embryonic medaka brain by whole-mount in situ hybridization using a probe for Apolipoprotein E (ApoE), a marker for activated microglia in teleosts. Three-dimensional imaging of the distribution of ApoE-expressing microglia in the irradiated embryonic brain clearly showed that ApoE-expressing microglia were abundant only in the late phase of phagocytosis during tissue repair induced by irradiation, while few microglia expressed ApoE in the initial phase of phagocytosis. This strongly suggests that ApoE has a significant function in the late phase of phagocytosis by microglia in the medaka brain. In addition, the distribution of microglia in p53-deficient embryos at the late phase of phagocytosis was almost the same as in wild-type embryos, despite the low numbers of irradiation-induced apoptotic neurons, suggesting that constant numbers of activated microglia were recruited at the late phase of phagocytosis irrespective of the extent of neuronal injury. This medaka model of microglia demonstrated specific recruitment after irradiation in the developing CNS and could provide a useful potential therapeutic strategy to counteract the detrimental effects of RT.     

Histological and Transcriptomic Analysis of Adult Japanese Medaka Sampled Onboard the International Space Station

To understand how humans adapt to the space environment, many experiments can be conducted on astronauts as they work aboard the Space Shuttle or the International Space Station (ISS). We also need animal experiments that can apply to human models and help prevent or solve the health issues we face in space travel. The Japanese medaka (Oryzias latipes) is a suitable model fish for studying space adaptation as evidenced by adults of the species having mated successfully in space during 15 days of flight during the second International Microgravity Laboratory mission in 1994. The eggs laid by the fish developed normally and hatched as juveniles in space. In 2012, another space experiment (“Medaka Osteoclast”) was conducted. Six-week-old male and female Japanese medaka (Cab strain osteoblast transgenic fish) were maintained in the Aquatic Habitat system for two months in the ISS. Fish of the same strain and age were used as the ground controls. Six fish were fixed with paraformaldehyde or kept in RNA stabilization reagent (n = 4) and dissected for tissue sampling after being returned to the ground, so that several principal investigators working on the project could share samples. Histology indicated no significant changes except in the ovary. However, the RNA-seq analysis of 5345 genes from six tissues revealed highly tissue-specific space responsiveness after a two-month stay in the ISS. Similar responsiveness was observed among the brain and eye, ovary and testis, and the liver and intestine. Among these six tissues, the intestine showed the highest space response with 10 genes categorized as oxidation–reduction processes (gene ontogeny term GO:0055114), and the expression levels of choriogenin precursor genes were suppressed in the ovary. Eleven genes including klf9, klf13, odc1, hsp70 and hif3a were upregulated in more than four of the tissues examined, thus suggesting common immunoregulatory and stress responses during space adaptation.     

Effects of serotonin on expression of the LDL receptor family member LR11 and 7-ketocholesterol-induced apoptosis in human vascular smooth muscle cells

Serotonin (5-HT) is a known mitogen for vascular smooth muscle cells (VSMCs). The dedifferentiation and proliferation/apoptosis of VSMCs in the arterial intima represent one of the atherosclerotic changes. LR11, a member of low-density lipoprotein receptor family, may contribute to the proliferation of VSMCs in neointimal hyperplasia. We conducted an in vitro study to investigate whether 5-HT is involved in LR11 expression in human VSMCs and apoptosis of VSMCs induced by 7-ketocholesterol (7KCHO), an oxysterol that destabilizes plaque. 5-HT enhanced the proliferation of VSMCs, and this effect was abolished by sarpogrelate, a selective 5-HT2A receptor antagonist. Sarpogrelate also inhibited the 5-HT-enhanced LR11 mRNA expression in VSMCs. Furthermore, 5-HT suppressed the 7KCHO-induced apoptosis of VSMCs via caspase-3/7-dependent pathway.