Parkin drives pS65‐Ub turnover independently of canonical autophagy in Drosophila

Parkinson''s disease‐related proteins, PINK1 and Parkin, act in a common pathway to maintain mitochondrial quality control. While the PINK1‐Parkin pathway can promote autophagic mitochondrial turnover (mitophagy) following mitochondrial toxification in cell culture, alternative quality control pathways are suggested. To analyse the mechanisms by which the PINK1–Parkin pathway operates in vivo, we developed methods to detect Ser65‐phosphorylated ubiquitin (pS65‐Ub) in Drosophila. Exposure to the oxidant paraquat led to robust, Pink1‐dependent pS65‐Ub production, while pS65‐Ub accumulates in unstimulated parkin‐null flies, consistent with blocked degradation. Additionally, we show that pS65‐Ub specifically accumulates on disrupted mitochondria in vivo. Depletion of the core autophagy proteins Atg1, Atg5 and Atg8a did not cause pS65‐Ub accumulation to the same extent as loss of parkin, and overexpression of parkin promoted turnover of both basal and paraquat‐induced pS65‐Ub in an Atg5‐null background. Thus, we have established that pS65‐Ub immunodetection can be used to analyse Pink1‐Parkin function in vivo as an alternative to reporter constructs. Moreover, our findings suggest that the Pink1‐Parkin pathway can promote mitochondrial turnover independently of canonical autophagy in vivo.     

Protective Role of miR-34c in Hypoxia by Activating Autophagy through BCL2 Repression

Hypoxia leads to significant cellular stress that has diverse pathological consequences such as cardiovascular diseases and cancers. MicroRNAs (miRNAs) are one of regulators of the adaptive pathway in hypoxia. We identified a hypoxia-induced miRNA, miR-34c, that was significantly upregulated in hypoxic human umbilical cord vein endothelial cells (HUVECs) and in murine blood vessels on day 3 of hindlimb ischemia (HLI). miR-34c directly inhibited BCL2 expression, acting as a toggle switch between apoptosis and autophagy in vitro and in vivo. BCL2 repression by miR-34c activated autophagy, which was evaluated by the expression of LC3-II. Overexpression of miR-34c inhibited apoptosis in HUVEC as well as in a murine model of HLI, and increased cell viability in HUVEC. Importantly, the number of viable cells in the blood vessels following HLI was increased by miR-34c overexpression. Collectively, our findings show that miR-34c plays a protective role in hypoxia, suggesting a novel therapeutic target for hypoxic and ischemic diseases in the blood vessels.     

Bovine liver dihydropyrimidine amidohydrolase: pH dependencies of inactivation by chelators and steady-state kinetic properties

Dihydropyrimidine amidohydrolase (EC 3.5.2.2) catalyzes the reversible hydrolysis of 5,6-dihydropyrimidines to the corresponding beta-ureido acids. Previous work has shown that incubation of this Zn2+ metalloenzyme with 2,6-dipicolinic acid, 8-hydroxyquinoline-5-sulfonic acid, or o-phenanthroline results in inactivation by Zn2+ removal by a reaction pathway involving formation of a ternary enzyme-Zn2+-chelator complex which subsequently dissociates to yield apoenzyme and the Zn2+-chelate (K. P. Brooks, E. A. Jones, B. D. Kim, and E. G. Sander, (1983) Arch. Biochem. Biophys. 226, 469-483). In the present work, the pH dependence of chelator inactivation is studied. The equilibrium constant for formation of the ternary complex is strongly pH dependent and increases with decreasing pH for all three chelators. There is a positive correlation between the value of the equilibrium constant observed for each chelator and the value of its stability constant for formation of Zn2+-chelate. The affinity of the chelators for the enzyme increases in the order 8-hydroxyquinoline-5-sulfonic acid greater than o-phenanthroline greater than 2,6-dipicolinic acid. The first-order rate constant for breakdown of the ternary complex to yield apoenzyme and Zn2+-chelate is invariant with pH for a given chelator but is different for each chelator, increasing in the reverse order. The pH dependence of the inactivation shows that two ionizable groups on the enzyme are involved in the inactivation. On the other hand, the steady-state kinetic behavior of the enzyme is well-described by ionization of a single group with a pK of 6.0 in the free enzyme. The basic form of the group is required for catalysis; protonation of the group decreases both Vmax and the apparent affinity for substrate. Conversely, binding of substrate decreases the pK of this group to about 5. L-Dihydroorotic acid is shown to be a competitive inhibitor of dihydropyrimidine amidohydrolase. Binding of L-dihydroorotic acid increases the pK of the ionizable group to 6.5. The agreement between the pK in the enzyme-L-dihydroorotic acid complex and the higher pK observed in the pH dependence of inactivation by chelators suggests that the same group is involved in the binding of acid, and chelators. The different effects of substrate and L-dihydroorotic acid on the pK suggest that the binding modes of these two ligands may be different and suggest a structural basis for the mutally exclusive substrate specificities of dihydropyrimidine amidohydrolase and dihydroorotase.     

The nucleotide sequence of a cDNA clone containing the entire coding region for mouse X-chromosome-linked phosphoglycerate kinase

A clone containing cDNA for X chromosome-linked phosphoglycerate kinase (PGK-1) was isolated from a mouse myeloma cDNA library. The nucleotide (nt) sequence of the cDNA has been determined, and the amino acid (aa) sequence of the enzyme thereby deduced. At the nt level, the coding region of mouse PGK cDNA has 93% homology with human X-linked cDNA and 60% homology with the yeast gene. Mouse PGK-1 protein contains 416 aa and is 98%, 96% and 64% homologous with human, horse, and yeast enzyme sequences, respectively.     

The two binding sites for DCMU in Photosystem II

Measurements on the fluorescence induction of Triton X-100 extracted Photosystem II (PSII) particles confirmed the existence of the two sites of inhibition in PSII for the herbicide DCMU. The two sites were located on the reducing and oxidizing sides of PSII, respectively. The inhibition on the oxidizing side, unlike that on the reducing side which was of the "none or all" type, was found only to slow down the electron donation at low concentrations of DCMU. The results also suggested that the inhibitions of DCMU at these two sites were mutually exclusive, i.e., the binding on one site prevented the binding on the other site.     

Computerized tomographic imaging of cryosurgical iceball formation in brain

Computerized tomography (CT) was used to monitor the exact anatomical location and dimensions of the cryosurgical iceball within the brain. The gross and microscopic appearance of the tissue iceball was examined in both acute and chronic animals. Iceball formation was monitored in the brain of four dogs under a general anesthesia. The radiographic image of the iceball was that of a well-demarcated radiolucent sphere that disappeared upon thawing. The post-thaw contrast-enhanced CT scan revealed a zone of blood-brain barrier breakdown extending no more than 1 mm beyond the maximum diameter that the iceball had attained. Histological examination demonstrated a sharp transition from frankly necrotic brain within the iceball to the normal cytoarchitecture of the surrounding neuropil. The safety and efficacy of a neurosurgical ablative procedure depends on the precision with which it can be generated. The use of CT imaging to monitor the formation of the cryosurgical iceball offers the neurosurgeon a means to precisely control the size of the ablative lesion. Small deeply situated brain tumors can be incorporated into the iceball under direct CT observation, thereby ensuring the completeness of the cryoablation while minimizing damage to the surrounding brain.