Oxidative stress–dependent phosphorylation activates ZNRF1 to induce neuronal/axonal degeneration

Oxidative stress is a well-known inducer of neuronal apoptosis and axonal degeneration. We previously showed that the E3 ubiquitin ligase ZNRF1 promotes Wallerian degeneration by degrading AKT to induce GSK3B activation. We now demonstrate that oxidative stress serves as an activator of the ubiquitin ligase activity of ZNRF1 by inducing epidermal growth factor receptor (EGFR)–mediated phosphorylation at the 103rd tyrosine residue and that the up-regulation of ZNRF1 activity by oxidative stress leads to neuronal apoptosis and Wallerian degeneration. We also show that nicotinamide adenine dinucleotide phosphate–reduced oxidase activity is required for the EGFR-dependent phosphorylation-induced activation of ZNRF1 and resultant AKT degradation via the ubiquitin proteasome system to induce Wallerian degeneration. These results indicate the pathophysiological significance of the EGFR–ZNRF1 pathway induced by oxidative stress in the regulation of neuronal apoptosis and Wallerian degeneration. A deeper understanding of the regulatory mechanism for ZNRF1 catalytic activity via phosphorylation will provide a potential therapeutic avenue for neurodegeneration.     

Detection of geosmin from <Emphasis Type="Italic">Coelosphaerium kuetzingianum</Emphasis> separated by a step density gradient medium from suspended materials in water in Lake Shinji,Japan

To identify the geosmin-producing organisms in Lake Shinji, Japan, we conducted preliminary chemical and biological investigations from April to July 2008, during which odor occurrence was observed. Geosmin was mainly detected in the suspended substance (SS) of lake water, and its concentration corresponded to the cell numbers of Coelosphaerium kuetzingianum (Cyanophyceae) and Monoraphidium contortum. C. kuetzingianum and Pseudodictyosphaerium minusculum (Chlorophyceae; which occurred only in April) were separated from the lake water of 30 April to the lighter, interlayers in a step density gradient medium. The highest concentration of geosmin was detected in the portion of the lightest layer separated from the water sample by gas chromatography mass spectrometry (GC/MS). Observation under optical microscope confirmed that abundant C. kuetzingianum with some P. minusculum and a very rare unknown material was retained in this portion. Similarly C. kuetzingianum was separated by the step density gradient medium from the sediment of 7 May. Abundant C. kuetzingianum with rare bacteria was recognized in the portion of the lightest layer, which contained the highest concentration of geosmin. The cell numbers of C. kuetzingianum correlated with the concentration of geosmin in the separated portions of water samples. These results suggest that C. kuetzingianum probably produced geosmin, although there has been no report about this to now.     

ZNRF1 promotes Wallerian degeneration by degrading AKT to induce GSK3B-dependent CRMP2 phosphorylation

Wallerian degeneration is observed in many neurological disorders, and it is therefore important to elucidate the axonal degeneration mechanism to prevent, and further develop treatment for, such diseases. The ubiquitin-proteasome system (UPS) has been implicated in Wallerian degeneration, but the underlying molecular mechanism remains unclear. Here we show that ZNRF1, an E3 ligase, promotes Wallerian degeneration by targeting AKT to degrade through the UPS. AKT phosphorylates glycogen synthase kinase-3β (GSK3B), and thereby inactivates it in axons. AKT overexpression significantly delays axonal degeneration. Overexpression of the active (non-phosphorylated) form of GSK3B induces CRMP2 phosphorylation, which is required for the microtubule reorganization observed in the degenerating axon. The inhibition of GSK3B and the overexpression of non-phosphorylated CRMP2 both protected axons from Wallerian degeneration. These findings indicate that the ZNRF1-AKT-GSK3B-CRMP2 pathway plays an important role in controlling Wallerian degeneration.     

Cy5-conjugated hybridization-sensitive fluorescent oligonucleotides for ratiometric analysis of nuclear poly(A)+ RNA

Subnuclear poly(A)(+) RNA localization in living mammalian cells was visualized by ratiometric analysis using hybridization-sensitive fluorescent oligonucleotide probes. Probes were oligonucleotides, which contained a Cy5 fluorescent dye at the strand end and a thiazole orange double-labeled nucleotide inside strand. A ratiometric analysis using poly(A)-targeting probes revealed a distribution of the probe itself as red fluorescence and localization of the target RNA sequence in cell nuclei as green fluorescence. The fluorescence of the subnuclear poly(A)(+) RNA hybridized with the poly(A)-targeting probes was observed as puncta in interchromatin areas.     

Exercise training augments the dynamic heart rate response to vagal but not sympathetic stimulation in rats

We examined the transfer function of autonomic heart rate (HR) control in anesthetized sedentary and exercise-trained (16 wk, treadmill for 1 h, 5 times/wk at 15 m/min and 15-degree grade) rats for comparison to HR variability assessed in the conscious resting state. The transfer function from sympathetic stimulation to HR response was similar between groups (gain, 4.2 ± 1.5 vs. 4.5 ± 1.5 beats·min(-1)·Hz(-1); natural frequency, 0.07 ± 0.01 vs. 0.08 ± 0.01 Hz; damping coefficient, 1.96 ± 0.55 vs. 1.69 ± 0.15; and lag time, 0.7 ± 0.1 vs. 0.6 ± 0.1 s; sedentary vs. exercise trained, respectively, means ± SD). The transfer gain from vagal stimulation to HR response was 6.1 ± 3.0 in the sedentary and 9.7 ± 5.1 beats·min(-1)·Hz(-1) in the exercise-trained group (P = 0.06). The corner frequency (0.11 ± 0.05 vs. 0.17 ± 0.09 Hz) and lag time (0.1 ± 0.1 vs. 0.2 ± 0.1 s) did not differ between groups. When the sympathetic transfer gain was averaged for very-low-frequency and low-frequency bands, no significant group effect was observed. In contrast, when the vagal transfer gain was averaged for very-low-frequency, low-frequency, and high-frequency bands, exercise training produced a significant group effect (P < 0.05 by two-way, repeated-measures ANOVA). These findings suggest that, in the frequency domain, exercise training augments the dynamic HR response to vagal stimulation but not sympathetic stimulation, regardless of the frequency bands.     

Review of acquired cystic disease-associated renal cell carcinoma with focus on pathobiological aspects

Acquired cystic disease (ACD)-associated renal cell carcinoma (RCC) is a recently established entity. In this article, we introduce the general view of this new entity. Macroscopically, the disease exclusively occurs in ACD and may arise as a dominant mass or non-dominant masses. Histologically, the tumor is characterized by a microcystic pattern, neoplastic cells with an eosinophilic or oncocytic cytoplasm and frequent intratumoral oxalate crystal deposition. Prominent nucleoli of tumor cells are often observed. Immunohistochemically, neoplastic cells are generally positive for AMACR but negative for cytokeratin 7. Ultrastructurally, neoplastic cells contain abundant mitochondria in the cytoplasm. Genetically, the gain of chromosomes 3, 7, 17 and abnormality of the sex chromosome were frequently observed in several studies. In conclusion, ACD-associated RCC may be widely recognized as a distinct entity in the near future because this tumor is morphologically and genetically different from other renal tumor entities that have been previously established.     

Telmisartan improves nonalcoholic steatohepatitis in medaka (Oryzias latipes) by reducing macrophage infiltration and fat accumulation

We investigated the efficacy of the antihypertensive drug telmisartan (Tel) and the mechanisms underlying the progression from simple steatosis to nonalcoholic steatohepatitis (NASH) in a medaka (Oryzias latipes) NASH model. We used the NASH activity score (NAS) developed in humans to assess the histology of the medaka NASH model and found that NAS increased with time. Further, TUNEL-positive apoptosis hepatocytes were found in the medaka NASH model. Tel administration resulted in the increased expression of liver peroxisome proliferator-activated receptor-??, carnitine palmitoyltransferase 1 and acyl-CoA oxidase 1 and decreased the number of 8-hydroxydeoxyguanosine-positive hepatocytes and the migration of macrophages positive for diastase-periodic-acid-Schiff. Medaka NAS was improved by Tel administration but fatty acid content was not affected. Tel reduced the infiltration of macrophages into the liver and ameliorated NASH pathology.     

Characterization of porcine autism susceptibility candidate 2 as a candidate gene for the number of corpora lutea in pigs

In a previous study, we mapped two quantitative trait loci (QTL) approximately 50cM apart, both influencing the number of corpora lutea in pigs on chromosome 3. One locus included functional candidate genes for proteins related to specific aspects of fertility, such as the follicle-stimulating hormone receptor and the luteinizing hormone/choriogonadotropin receptor. However, specific genes related to the second locus have not yet been identified. This study aims to identify another candidate gene influencing the number of corpora lutea in pigs. Using 12 polymorphic markers, we fine-mapped a narrow region of pig chromosome 3 that had been shown to contain a QTL for corpora lutea. In the critical region, only 1 gene, autism susceptibility candidate 2 (AUTS2), was identified as a positional candidate. Our results demonstrate that the porcine AUTS2 gene consists of 19 exons with a complete open reading frame of 3768bp encoding an AUTS2 protein of 1256 amino acids. We screened the whole coding sequence and parts of the untranslated region for polymorphisms in an F(2) population of Duroc×Meishan crosses. We found 1 ins/del and 7 single nucleotide polymorphisms (SNP), including 2 nonsynonymous variants, c.943C>T in exon 7 and c.2828C>T in exon 19, resulting in P315S and A943V, respectively. The SNP c.943C>T within a proline-rich domain was genotyped in several breeds; the C allele occurred in all breeds, whereas the T allele occurred only in Meishan pigs. Using in situ hybridization, the mRNA expression of the AUTS2 gene was observed on granulosa cells in the porcine ovary and thus may be associated with hormone sensitivity.