Gray and Red Fragments of the Egg of the Ascidian Ciona savignyi: Preferential Development of Muscle Cells from Gray Fragments

The egg of the ascidian Ciona savignyi is pinkish red with brownish myoplasm that contains the putative determinants responsible for differentiation of muscle cells. When dechorionated unfertilized eggs were centrifuged at moderate speed, eggs were divided into centripetal, small gray fragments and centrifugal, large red fragments. The former contained the female pronucleus and clear cytoplasm, while most of the latter was filled with yolk granules. An antibody raised against the myoplasm of C. intestinalis eggs extensively stained the cortical region of gray fragments, while the antibody stained only small regions of the red fragments. After insemination, both fragments cleaved and gave rise to partial embryos. When development of muscle and epidermal cells in the partial embryos was examined with specific antibodies, muscle development was conspicuous in gray partial embryos, while epidermal differentiation was extensive in red partial embryos. Furthermore, when expression of markers of differentiation was examined in cleavage-arrested gray and red fragments, the number of arrested gray fragments exhibiting the muscle marker was about three-fold greater than in controls. These results suggest that putative muscle determinants are concentrated into gray fragments.     

Ammonia induces amyloidogenesis in astrocytes by promoting amyloid precursor protein translocation into the endoplasmic reticulum

Hyperammonemia is known to cause various neurological dysfunctions such as seizures and cognitive impairment. Several studies have suggested that hyperammonemia may also be linked to the development of Alzheimer’s disease (AD). However, the direct evidence for a role of ammonia in the pathophysiology of AD remains to be discovered. Herein, we report that hyperammonemia increases the amount of mature amyloid precursor protein (mAPP) in astrocytes, the largest and most prevalent type of glial cells in the central nervous system that are capable of metabolizing glutamate and ammonia, and promotes amyloid beta (Aβ) production. We demonstrate the accumulation of mAPP in astrocytes was primarily due to enhanced endocytosis of mAPP from the plasma membrane. A large proportion of internalized mAPP was targeted not to the lysosome, but to the endoplasmic reticulum, where processing enzymes β-secretase BACE1 (beta-site APP cleaving enzyme 1) and γ-secretase presenilin-1 are expressed, and mAPP is cleaved to produce Aβ. Finally, we show the ammonia-induced production of Aβ in astrocytic endoplasmic reticulum was specific to Aβ42, a principal component of senile plaques in AD patients. Our studies uncover a novel mechanism of Aβ42 production in astrocytes and also provide the first evidence that ammonia induces the pathogenesis of AD by regulating astrocyte function.     

Mechanism for generation of hydrogen peroxide and change of mitochondrial membrane potential during rotenone-induced apoptosis

Rotenone, an inhibitor of NADH dehydrogenase complex, is a naturally occurring insecticide, which is capable of inducing apoptosis. Rotenone-induced apoptosis is considered to contribute to its anticancer effect and the etiology of Parkinson's disease (PD). We demonstrated that rotenone induced internucleosomal DNA fragmentation, DNA ladder formation, in human cultured cells, HL-60 (promyelocytic leukemia) and BJAB cells (B-cell lymphoma). Flow cytometry showed that rotenone induced H2O2 generation, followed by significant changes in the mitochondrial membrane potential (DeltaPsim). Caspase-3 activity increased in HL-60 cells in a time-dependent manner. These apoptotic events were delayed in HP100 cells, an H2O2-resistant clone of HL-60, confirming the involvement of H2O2 in apoptosis. Expression of anti-apoptotic protein, Bcl-2, in BJAB cells drastically inhibited DeltaPsim change and DNA ladder formation but not H2O2 generation, confirming the participation of mitochondrial dysfunction in apoptosis. NAD(P)H oxidase inhibitors prevented H2O2 generation and DNA ladder formation. These results suggest that rotenone induces O2(-)-derived H2O2 generation through inhibition of NADH dehydrogenase complex and/or activation of NAD(P)H oxidase, and H2O2 generation causes the disruption of mitochondrial membrane in rotenone-induced apoptosis.     

Aberrant Active cis-Regulatory Elements Associated with Downregulation of RET Finger Protein Overcome Chemoresistance in Glioblastoma

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Parkin stabilizes PINK1 through direct interaction

Parkinson disease (PD) is the most common movement disorder and is characterized by dopaminergic dysfunction. The majority of PD cases are sporadic; however, the discovery of genes linked to rare familial forms of the disease has provided crucial insight into the molecular mechanisms of disease pathogenesis. Multiple genes mediating familial forms of Parkinson’s disease (PD) have been identified, such as parkin (PARK2) and phosphatase and tensin homologue deleted on chromosome ten (PTEN)-induced putative kinase 1: PINK1 (PARK6). Here, we showed that Parkin directly interacts with PINK1, but did not bind to pathogenic PINK1 mutants. Parkin, but not its pathogenic mutants, stabilizes PINK1 by interfering with its degradation via the ubiquitin-mediated proteasomal pathway. In addition, the interaction between Parkin and PINK1 resulted in reciprocal reduction of their solubility. Our results indicate that Parkin regulates PINK1 stabilization via direct interaction with PINK1, and operates through a common pathway with PINK1 in the pathogenesis of early-onset PD.     

Processing of DNA lesions by archaeal DNA polymerases from Sulfolobus solfataricus

Spontaneous damage to DNA as a result of deamination, oxidation and depurination is greatly accelerated at high temperatures. Hyperthermophilic microorganisms constantly exposed to temperatures exceeding 80°C are endowed with powerful DNA repair mechanisms to maintain genome stability. Of particular interest is the processing of DNA lesions during replication, which can result in fixed mutations. The hyperthermophilic crenarchaeon Sulfolobus solfataricus has two functional DNA polymerases, PolB1 and PolY1. We have found that the replicative DNA polymerase PolB1 specifically recognizes the presence of the deaminated bases hypoxanthine and uracil in the template by stalling DNA polymerization 3–4 bases upstream of these lesions and strongly associates with oligonucleotides containing them. PolB1 also stops at 8-oxoguanine and is unable to bypass an abasic site in the template. PolY1 belongs to the family of lesion bypass DNA polymerases and readily bypasses hypoxanthine, uracil and 8-oxoguanine, but not an abasic site, in the template. The specific recognition of deaminated bases by PolB1 may represent an initial step in their repair while PolY1 may be involved in damage tolerance at the replication fork. Additionally, we reveal that the deaminated bases can be introduced into DNA enzymatically, since both PolB1 and PolY1 are able to incorporate the aberrant DNA precursors dUTP and dITP.     

Binding and transport of SFPQ-RNA granules by KIF5A/KLC1 motors promotes axon survival

Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long-distance transport of RNA granules is not yet understood. Here, we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts selectively with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that the binding of SFPQ to the KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie Tooth (CMT) disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations indicate that KIF5A-mediated SFPQ-RNA granule transport may be a key function disrupted in KIF5A-linked neurologic diseases and that replacing axonally translated proteins serves as a therapeutic approach to axonal degenerative disorders.     

Secretory and continuous expression of Aspergillus niger glucose oxidase gene in Pichia pastoris

We proposed a yeast transformant cell incorporating the Aspergillus niger glucose oxidase gene (GOX gene), which is capable of constitutively as well as secretory expression. The GOX gene has been cloned in this study. This conclusion is based on the following: first, the ligated DNA determined by electrophoresis, was a 1489-1882bp fragment, close to the size of glucose oxidase (GOD), which is 1818bp. Secondly, the single open reading frame encoded a protein of 605 amino acids. Thirdly, secreted GOD recombinant proteins in the culture supernatants of the GOX gene transformant migrated as a single band in SDS-PAGE with an apparent molecular mass of between 75,000 and 100,000 Da, which is glycosylated GOD by the Pichia pastoris X-33 host machinery during the secretion process. Finally, the clones were cultured and secreted a protein, which possessed the GOD activity of catalyzing beta-d-glucose oxidation. With regard to the pH characteristics, the activity was more than 80% of the maximum activity in the range between pH 5 and pH 7. As for the temperature characteristics, the activity was not less than 92% of the maximum in the temperature range between 10 and 45 degrees C. The GOX gene transformant was able to maintain the GOD enzyme activity and produce recombinant GOD continuously for at least 2 weeks.