Ablation of Vacuole Protein Sorting 18 (Vps18) Gene Leads to Neurodegeneration and Impaired Neuronal Migration by Disrupting Multiple Vesicle Transport Pathways to Lysosomes

Intracellular vesicle transport pathways are critical for neuronal survival and central nervous system development. The Vps-C complex regulates multiple vesicle transport pathways to the lysosome in lower organisms. However, little is known regarding its physiological function in mammals. We deleted Vps18, a central member of Vps-C core complex, in neural cells by generating Vps18^F/F; Nestin-Cre mice (Vps18 conditional knock-out mice). These mice displayed severe neurodegeneration and neuronal migration defects. Mechanistic studies revealed that Vps18 deficiency caused neurodegeneration by blocking multiple vesicle transport pathways to the lysosome, including autophagy, endocytosis, and biosynthetic pathways. Our study also showed that ablation of Vps18 resulted in up-regulation of β1 integrin in mouse brain probably due to lysosome dysfunction but had no effects on the reelin pathway, expression of N-cadherin, or activation of JNK, which are implicated in the regulation of neuronal migration. Finally, we demonstrated that knocking down β1 integrin partially rescued the migration defects, suggesting that Vps18 deficiency-mediated up-regulation of β1 integrin may contribute to the defect of neuronal migration in the Vps18-deficient brain. Our results demonstrate important roles of Vps18 in neuron survival and migration, which are disrupted in multiple neural disorders.     

Distinct acyl protein transferases and thioesterases control surface expression of calcium-activated potassium channels

Protein palmitoylation is rapidly emerging as an important determinant in the regulation of ion channels, including large conductance calcium-activated potassium (BK) channels. However, the enzymes that control channel palmitoylation are largely unknown. Indeed, although palmitoylation is the only reversible lipid modification of proteins, acyl thioesterases that control ion channel depalmitoylation have not been identified. Here, we demonstrate that palmitoylation of the intracellular S0-S1 loop of BK channels is controlled by two of the 23 mammalian palmitoyl-transferases, zDHHC22 and zDHHC23. Palmitoylation by these acyl transferases is essential for efficient cell surface expression of BK channels. In contrast, depalmitoylation is controlled by the cytosolic thioesterase APT1 (LYPLA1), but not APT2 (LYPLA2). In addition, we identify a splice variant of LYPLAL1, a homolog with ～30% identity to APT1, that also controls BK channel depalmitoylation. Thus, both palmitoyl acyltransferases and acyl thioesterases display discrete substrate specificity for BK channels. Because depalmitoylated BK channels are retarded in the trans-Golgi network, reversible protein palmitoylation provides a critical checkpoint to regulate exit from the trans-Golgi network and thus control BK channel cell surface expression.     

Diosgenin induces apoptosis in IGF-1-stimulated human thyrocytes through two caspase-dependent pathways

Insulin-like growth factor-1 (IGF-1) is a growth factor of the thyroid that has been shown in our previous study to possess proliferative and antiapoptotic effects in FRTL-5 cell lines through the upregulation of cyclin D and Fas-associated death domain-like interleukin-1-converting enzyme (FLICE)-inhibitory protein (FLIP). Diosgenin, a natural steroid sapogenin from plants, has been shown to induce apoptosis in many cell lines, with the exception of thyroid cells. In this report, we investigated the apoptotic effect and mechanism of diosgenin in IGF-1-stimulated primary human thyrocytes. Primary human thyrocytes were preincubated with or without IGF-1 for 24h and subsequently exposed to varying concentrations of diosgenin for different times. We found that diosgenin induced apoptosis in human thyrocytes pretreated with IGF-1 in a dose-dependent manner through the activation of caspase cascades. Moreover, diosgenin inhibited FLIP and activated caspase-8 in the FAS-related apoptotic pathway. Diosgenin increased the production of ROS, regulated the balance of Bax and Bcl-2 and cleaved caspase-9 in the mitochondrial apoptotic pathway. These results indicate that diosgenin induces apoptosis in IGF-1-stimulated primary human thyrocytes through two caspase-dependent pathways.     

iASPPsv antagonizes apoptosis induced by chemotherapeutic agents in MCF-7 cells and mouse thymocytes

iASPP was an inhibitory member of ASPP family and could specifically inhibit the apoptotic function of p53. iASPPsv was identified by our lab as the short isoform of iASPP, which encoded a 407aa protein and highly matched the carboxyl terminus of iASPP. In this study, iASPPsv was stably transfected into the breast cancer cell line MCF-7 by means of lentivirus to explore the effects of iASPPsv on biological functions of MCF-7. Thymocytes from iASPP/iASPPsv transgenic mice were also used to explore the effects of iASPP/iASPPsv on cell biological function. The results demonstrated that iASPPsv antagonized the growth inhibition induced by etoposide (VP-16) in MCF-7 cells. iASPPsv also down-regulated proapoptotic genes (Bax, Puma and Noxa) expression to inhibit apoptosis caused by VP-16. Moreover, iASPP and iASPPsv could both help the thymocytes of transgenic mice to resist the growth inhibition and apoptosis caused by dexamethasone (Dex) or VP-16. At the same time, DNA double strand break damage accumulated in either iASPPsv MCF-7 cells or iASPP/iASPPsv thymocytes. These findings showed that iAPSS/iASPPsv reduced the growth inhibition and apoptosis induced by Dex or VP-16, with DNA damage accumulating which might promote the pathogenesis and/or progression of cancer.     

Characteristic morphology of intracellular microcolonies of Legionella oakridgensis OR-10

Legionella oakridgensis occasionally causes pneumonia in humans. We report here the characteristic morphology of intracellular microcolonies of L. oakridgensis OR-10 in infected epithelial cells. By light microscopy after Gimenez staining, the bacteria showed serpentine-like chain, disk-like conglomerate, and granular forms when they grew intracellularly in Vero cells, HeLa cells, and A549 cells. In a time-lapse study, we observed the progressive change from a serpentine-like chain form to a conglomerate form in Vero cells. Transmission electron microscopy showed that L. oakridgensis OR-10 proliferated both inside membrane structures and in the cytoplasm. Such highly serpentine chain growth has not been reported in any intracellular bacteria. Furthermore, these results imply that L. oakridgensis OR-10 may be proliferating inside the endoplasmic reticulum.     

Ezrin functionality and hypothermic preservation injury in LLC-PK1 cells

Renal epithelial cells from donor kidneys are susceptible to hypothermic preservation injury, which is attenuated when they over express the cytoskeletal linker protein ezrin. This study was designed to characterize the mechanisms of this protection. Renal epithelial cell lines were created from LLC-PK1 cells, which expressed mutant forms of ezrin with site directed alterations in membrane binding functionality. The study used cells expressing wild type ezrin, T567A, and T567D ezrin point mutants. The A and D mutants have constitutively inactive and active membrane binding conformations, respectively. Cells were cold stored (4 °C) for 6-24 h and reperfused for 1h to simulate transplant preservation injury. Preservation injury was assessed by mitochondrial activity (WST-1) and LDH release. Cells expressing the active ezrin mutant (T567D) showed significantly less preservation injury compared to wild type or the inactive mutant (T567A), while ezrin-specific siRNA knockdown and the inactive mutant potentiated preservation injury. Ezrin was extracted and identified from purified mitochondria. Furthermore, isolated mitochondria specifically bound anti-ezrin antibodies, which were reversed with the addition of exogenous recombinant ezrin. Recombinant wild type ezrin significantly reduced the sensitivity of the mitochondrial permeability transition pore (mPTP) to calcium, suggesting ezrin may modify mitochondrial function. In conclusion, the cytoskeletal linker protein ezrin plays a significant role in hypothermic preservation injury in renal epithelia. The mechanisms appear dependent on the molecule's open configuration (traditional linker functionality) and possibly a novel mitochondrial specific role, which may include modulation of mPTP function or calcium sensitivity.     

The effect of total spinal anesthesia on cardiac function in a large animal model of brain death

Brain death (BD) causes cardiac dysfunction in organ donors, attributable to the catecholamine storm that occurs with raised intracerebral pressure (ICP). However the direct contribution of the spinal sympathetics has not been well described. We examined the effect of total spinal anesthesia (TSA) on cardiac function in a large animal model of BD. Eighteen pigs were allocated to 3 experimental groups: Group?1, the saline-treated control group; Group?2, TSA administered prior to BD; and Group?3, TSA administered 30?min after BD. Inflation of an intracerebral balloon-tipped catheter was used to induce BD. Ventricular function was assessed using a pressure-volume loop catheter and magnetic resonance imaging. Serum catecholamine levels were assessed with high performance liquid chromatography. Inflation of the intracerebral balloon-tipped catheter was associated with a dramatic rise in heart rate and blood pressure, along with increased concentrations of serum epinephrine and norepinephrine. This phenomenon was not observed in Group 2. In Group 1, there was a significant decline in contractility, whereas groups 2 and?3 saw no change. Group 2 had greater contractile reserve than groups 1 and 3. Our data demonstrate the central role of spinal sympathetics in the hemodynamic response to raised ICP. Further work is required to determine the utility of TSA in reversing cardiac dysfunction in BD donors.     

Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro

Hypoxia is a common feature of solid tumors and an important contributor to tumor radioresistance. miR-210 is the most consistently and robustly induced microRNA under hypoxia in different types of tumor cells and normal cells. In the present study, to explore the feasibility of miR-210 as an effective therapeutic target, lentiviral-mediated anti-sense miR-210 gene transfer technique was employed to downregulate miR-210 expression in hypoxic human hepatoma SMMC-7721, HepG2 and HuH7 cells, and phenotypic changes of which were analyzed. Hypoxia led to an increased hypoxia inducible factor-1α (HIF-1α) and miR-210 expression and cell arrest in the G(0)/G(1) phase in all cell lines. miR-210 downregulation significantly suppressed cell viability, induced cell arrest in the G(0)/G(1) phase, increased apoptotic rate and enhanced radiosensitivity in hypoxic human hepatoma cells. Moreover, apoptosis-inducing factor, mitochondrion-associated, 3 (AIFM3) was identified as a direct target gene of miR-210. AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells. Taken together, these data suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy might be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells.     

A novel porcine circovirus type 2a strain, 10JS-2, with eleven-nucleotide insertions in the origin of genome replication

Here, we report a novel porcine circovirus type 2a (PCV2a) strain with 11 nucleotides (nt) inserted in the origin of genome replication (Ori). This is the first report of a PCV2a strain with nucleotide insertion in Ori. Our study will help further epidemiological studies and extend our knowledge of evolutionary characteristics of PCV2.     

Complete Genome Sequence of a Porcine Orthoreovirus from Southern China

Porcine orthoreoviruses belong to the family Reoviridae and cause mainly mild enteritis in piglets. We present here the complete genome sequence of a novel porcine orthoreovirus strain (GD-1) isolated from a piglet in southern China. Our data will facilitate future investigations of the molecular characteristics and epidemiology of porcine orthoreoviruses.