SOD1 (copper/zinc superoxide dismutase) deficiency drives amyloid β protein oligomerization and memory loss in mouse model of Alzheimer disease

Oxidative stress is closely linked to the pathogenesis of neurodegeneration. Soluble amyloid β (Aβ) oligomers cause cognitive impairment and synaptic dysfunction in Alzheimer disease (AD). However, the relationship between oligomers, oxidative stress, and their localization during disease progression is uncertain. Our previous study demonstrated that mice deficient in cytoplasmic copper/zinc superoxide dismutase (CuZn-SOD, SOD1) have features of drusen formation, a hallmark of age-related macular degeneration (Imamura, Y., Noda, S., Hashizume, K., Shinoda, K., Yamaguchi, M., Uchiyama, S., Shimizu, T., Mizushima, Y., Shirasawa, T., and Tsubota, K. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 11282-11287). Amyloid assembly has been implicated as a common mechanism of plaque and drusen formation. Here, we show that Sod1 deficiency in an amyloid precursor protein-overexpressing mouse model (AD mouse, Tg2576) accelerated Aβ oligomerization and memory impairment as compared with control AD mouse and that these phenomena were basically mediated by oxidative damage. The increased plaque and neuronal inflammation were accompanied by the generation of N(ε)-carboxymethyl lysine in advanced glycation end products, a rapid marker of oxidative damage, induced by Sod1 gene-dependent reduction. The Sod1 deletion also caused Tau phosphorylation and the lower levels of synaptophysin. Furthermore, the levels of SOD1 were significantly decreased in human AD patients rather than non-AD age-matched individuals, but mitochondrial SOD (Mn-SOD, SOD2) and extracellular SOD (CuZn-SOD, SOD3) were not. These findings suggest that cytoplasmic superoxide radical plays a critical role in the pathogenesis of AD. Activation of Sod1 may be a therapeutic strategy for the inhibition of AD progression.     

Development of Ca²(+) signaling mechanisms and cell motility in presumptive ectodermal cells during amphibian gastrulation

This study investigated the development of Ca2(+) signaling mechanisms and their role in initiating morphogenetic cell movement in the presumptive ectoderm of Japanese newt (Cynops pyrrhogaster) during gastrulation. Histochemical staining using fluorescently labeled ryanodine and dihydropyridine probes revealed that dihydropyridine receptor (L-type Ca2(+) channels) appeared in stage 12b embryos, while ryanodine receptors were expressed in both stage 11 and 12b embryos. Transmission electron microscopy of stage 12b embryos showed abundant peripheral couplings, which are couplings of the endoplasmic reticulum and cell membrane with an approximate 12 nm gap. Caffeine increased the intracellular free Ca2(+) concentration ([Ca2(+)](i)) in presumptive ectodermal cells isolated from both stage 11 and 12b embryos, while (±)-Bay K 8644 ((±)-BayK) increased [Ca2(+)](i) in cells isolated from stage 12b embryos, but not in cells isolated from stage 11 embryos. Dantrolene and nifedipine completely inhibited increases in [Ca2(+)](i) after treatment with caffeine and (±)-BayK, respectively. Caffeine activated the motility of cells isolated from both stage 11 and 12b embryos, but (±)-BayK only activated the motility of cells isolated from stage 12b embryos. These findings suggested that formation of the Ca2(+) -induced Ca2(+) release system in presumptive ectodermal cells during gastrulation plays an important role in the initiation and execution of epibolic extension.     

Isolation and characterization of a barley yellow stripe-like gene, HvYSL5

Yellow stripe-like (YSL) family transporters, belonging to a novel subfamily of oligopeptide transporter (OPT), has been proposed to be involved in metal uptake and long-distance transport, but only a few of them have been functionally characterized so far. In the present study, we isolated an uncharacterized member of the YSL family, HvYSL5, in barley based on expressed sequence tag (EST) information. HvYSL5 shared 50% identity with HvYS1, a transporter for the ferric-mugineic acid complex, at the amino acid level. Promoter analysis showed that the HvYSL5 upstream sequence contains both iron deficiency response element 1 and 2 (IDE1 and 2). HvYSL5 was expressed in the roots and the expression was greatly induced by Fe deficiency, but not by deficiency of other metals including Zn, Cu and Mn. Spatial investigation showed that much higher expression of HvYSL5 was found in the mature zones of the roots, but not in the root tips. Furthermore, the expression showed a diurnal rhythm, being the highest in the morning, but with no expression in the afternoon. HvYSL5 was localized in all root cells, and subcellular localization analysis showed that HvYSL5 is likely to be localized in the vesicles. Knockdown of HvYSL5 did not result in any detectable phenotype changes. Although the exact role of HvYSL5 remains to be examined, our results suggest that it is involved in the transient storage of Fe or phytosiderophores.     

Global gene expression profiling in early-stage polycystic kidney disease in the Han:SPRD Cy rat identifies a role for RXR signaling

Han:SPRD Cy is a spontaneous rat model of polycystic kidney disease (PKD) caused by a missense mutation in Pkdr1. Cystogenesis in this model is not clearly understood. In the current study, we performed global gene expression profiling in early-stage PKD cyst development in Cy/Cy kidneys and normal (+/+) kidneys at 3 and 7 days of postnatal age. Expression profiles were determined by microarray analysis, followed by validation with real-time RT-PCR. Genes were selected with over 1.5-fold expression changes compared with age-matched +/+ kidneys for canonical pathway analysis. We found nine pathways in common between 3- and 7-day Cy/Cy kidneys. Three significantly changed pathways were designated "Vitamin D Receptor (VDR)/Retinoid X Receptor (RXR) Activation," "LPS/IL-1-Mediated Inhibition of RXR Function," and "Liver X Receptor (LXR)/RXR Activation." These results suggest that RXR-mediated signaling is significantly altered in developing kidneys with mutated Pkdr1. In gene ontology analysis, the functions of these RXR-related genes were found to be involved in regulating cell proliferation and organ morphogenesis. With real-time RT-PCR analysis, the upregulation of Ptx2, Alox15b, OSP, and PCNA, major markers of cell proliferation associated with the RXR pathway, were confirmed in 3- and 7-day Cy/Cy kidneys compared with 3-day +/+ kidneys. The increased RXR protein was observed in both the nucleus and cytoplasm of cystic epithelial cells in early-stage Cy/Cy kidneys, and the RXR-positive cells were strongly positive for PCNA staining. Taken together, cell proliferation and organ morphogenesis signals transduced by RXR-mediated pathways may have important roles for cystogenesis in early-stage PKD in this Pkdr1-mutated Cy rat.     

Deduction of the evaluation limit and termination timing of multi-round protein misfolding cyclic amplification from a titration curve

In this study, the efficacy of disinfectants in reducing the partially protease-resistant isoform of prion protein was evaluated by a multi-round protein misfolding cyclic amplification (PMCA) technique. Hamster brains infected with scrapie-derived strain 263K were homogenized, treated under inactivating or mock conditions, and subjected to multi-round PMCA. Four sets of serial 10-fold dilutions of mock-treated samples were analyzed. Although considerable variability was observed in the signal patterns, between the second and sixth rounds the number of the PMCA round correlated in a linear fashion with the mean dilution factor of mock-treated, infected brains, corresponding to a log reduction factor (LRF) of 3.8-7.3 log. No signals were observed in the PMCA products amplified from normal hamster brain homogenates. The mean numbers of rounds at the first appearance of the signal for 1 M and 2 M NaOH-treated samples were 4.33 and 4, respectively. Using the linear regression line as the titration curve, the LRFs of these disinfectants were found to be 6.1 and 5.8 log, respectively; these values were not significantly different. The mean number of rounds for the alkaline cleaner and sodium dodecyl sulfate were 9 and 10.33, respectively, and were outside the range of both the linear regression line and evaluation limit. The disinfectants were considered very effective because their LRFs were ≥7.3 log. These estimations were concordant with previous bioassay-based reports. Thus, the evaluation limit seems to be valuable in some applications of multi-round PMCA, such as disinfectant assessment and process validation.     

Interaction between vestibulo-cardiovascular reflex and arterial baroreflex during postural change in rats

To examine a cooperative role for the baroreflex and the vestibular system in controlling arterial pressure (AP) during voluntary postural change, AP was measured in freely moving conscious rats, with or without sinoaortic baroreceptor denervation (SAD) and/or peripheral vestibular lesion (VL). Voluntary rear-up induced a slight decrease in AP (-5.6 ± 0.8 mmHg), which was significantly augmented by SAD (-14.7 ± 1.0 mmHg) and further augmented by a combination of VL and SAD (-21 ± 1.0 mmHg). Thus we hypothesized that the vestibular system sensitizes the baroreflex during postural change. To test this hypothesis, open-loop baroreflex analysis was conducted on anesthetized sham-treated and VL rats. The isolated carotid sinus pressure was increased stepwise from 60 to 180 mmHg while rats were placed horizontal prone or in a 60° head-up tilt (HUT) position. HUT shifted the carotid sinus pressure-sympathetic nerve activity (SNA) relationship (neural arc) to a higher SNA, shifted the SNA-AP relationship (peripheral arc) to a lower AP, and, consequently, moved the operating point to a higher SNA while maintaining AP (from 113 ± 5 to 114 ± 5 mmHg). The HUT-induced neural arc shift was completely abolished in VL rats, whereas the peripheral arc shifted to a lower AP and the operating point moved to a lower AP (from 116 ± 3 to 84 ± 5 mmHg). These results indicate that the vestibular system elicits sympathoexcitation, shifting the baroreflex neural arc to a higher SNA and maintaining AP during HUT.     

Microbial diversity with dominance of 16S rRNA gene sequences with high GC contents at 74 and 98 °C subsurface crude oil deposits in Japan

We prepared DNA from the production waters of oil deposits and wellheads of the high- and hypertemperature Japanese oil wells #AR39 (depth, 1230 m; temperature, 74 °C; pressure, 2.92 MPa) and #SR123 (depth, 1687 m; temperature, 98 °C; pressure, 11.3 MPa) to detect indigenous bacterial and archaeal microorganisms. We used PCR to amplify the 16S rRNA genes of microbial communities and characterized them based on their sequences. A few species of microorganisms with high GC contents were detected in samples from oil deposits, whereas the microbial constituents and their GC contents were diverse in wellhead samples. A comparison of the composition of the microbial communities found that the predominant indigenous populations in the #SR123 oil deposit were Thermotoga hypogea-, Thermotoga petrophila- and Thermodesulfobacterium commune-like bacteria with a 61-63% GC content in their 16S rRNA gene sequences, and Archaeoglobus fulgidus-like archaea with a 65% GC content, whereas the major population in #AR39 comprised Thermacetogenium phaeum- and Fervidobacterium pennavorans-like bacteria and Methanothermobacter thermautotrophicus-like archaea with a 60%, 60% and 61% GC content, respectively.