Mechanisms Underlying Plk1 Polo-Box Domain-Mediated Biological Processes and Their Physiological Significance

Mammalian polo-like kinase 1 (Plk1) has been studied intensively as a key regulator of various cell cycle events that are critical for proper M-phase progression. The polo-box domain (PBD) present in Plk1’s C-terminal non-catalytic region has been shown to play a central role in targeting the N-terminal kinase domain of Plk1 to specific subcellular locations. Subsequent studies reveal that PBD binds to a phosphorylated motif generated by one of the two mechanisms - self-priming by Plk1 itself or non-self-priming by a Pro-directed kinase, such as Cdc2. Here, we comparatively review the differences in the biochemical steps of these mechanisms and discuss their physiological significance. Considering the diverse functions of Plk1 during the cell cycle, a better understanding of how the catalytic activity of Plk1 functions in concert with its cis-acting PBD and how this coordinated process is intricately regulated to promote Plk1 functions will be important for providing new insights into different mechanisms underlying various Plk1-mediated biological events that occur at the multiple stages of the cell cycle.     

Inhibition of GM3 Synthase Attenuates Neuropathology of Niemann-Pick Disease Type C by Affecting Sphingolipid Metabolism

In several lysosomal storage disorders, including Niemann-Pick disease Type C (NP-C), sphingolipids, including glycosphingolipids, particularly gangliosides, are the predominant storage materials in the brain, raising the possibility that accumulation of these lipids may be involved in the NP-C neurodegenerative process. However, correlation of these accumulations and NP-C neuropathology has not been fully characterized. Here we derived NP-C mice with complete and partial deletion of the Siat9 (encoding GM3 synthase) gene in order to investigate the role of ganglioside in NP-C pathogenesis. According to our results, NPC mice with homozygotic deletion of GM3 synthase exhibited an enhanced neuropathological phenotype and died significantly earlier than NP-C mice. Notably, in contrast to complete depletion, NP-C mice with partial deletion of the GM3 synthase gene showed ameliorated NP-C neuropathology, including motor disability, demyelination, and abnormal accumulation of cholesterol and sphingolipids. These findings indicate the crucial role of GM3 synthesis in the NP-C phenotype and progression of CNS pathologic abnormality, suggesting that well-controlled inhibition of GM3 synthesis could be used as a therapeutic strategy.     

The jiaoyao1 Mutant Is an Allele of korrigan1 That Abolishes Endoglucanase Activity and Affects the Organization of Both Cellulose Microfibrils and Microtubules in Arabidopsis

In higher plants, cellulose is synthesized by plasma membrane–localized cellulose synthase complexes (CSCs). Arabidopsis thaliana GH9A1/KORRIGAN1 is a membrane-bound, family 9 glycosyl hydrolase that is important for cellulose synthesis in both primary and secondary cell walls. Most previously identified korrigan1 mutants show severe phenotypes such as embryo lethality; therefore, the role of GH9A1 in cellulose synthesis remains unclear. Here, we report a novel A577V missense mutation, designated jiaoyao1 (jia1), in the second of the glycosyl hydrolase family 9 active site signature motifs in GH9A1. jia1 is defective in cell expansion in dark-grown hypocotyls, roots, and adult plants. Consistent with its defect in cell expansion, this mutation in GH9A1 resulted in reduced cellulose content and reduced CSC velocity at the plasma membrane. Green fluorescent protein–GH9A1 is associated with CSCs at multiple locations, including the plasma membrane, Golgi, trans-Golgi network, and small CESA-containing compartments or microtubule-associated cellulose synthase compartments, indicating a tight association between GH9A1 and CSCs. GH9A1^A577V abolishes the endoglucanase activity of GH9A1 in vitro but does not affect its interaction with CESAs in vitro, suggesting that endoglucanase activity is important for cellulose synthesis. Interestingly, jia1 results in both cellulose microfibril and microtubule disorganization. Our study establishes the important role of endoglucanase in cellulose synthesis and cellulose microfibril organization in plants.     

Persistent telomere cohesion triggers a prolonged anaphase

Telomeres use distinct mechanisms (not used by arms or centromeres) to mediate cohesion between sister chromatids. However, the motivation for a specialized mechanism at telomeres is not well understood. Here we show, using fluorescence in situ hybridization and live-cell imaging, that persistent sister chromatid cohesion at telomeres triggers a prolonged anaphase in normal human cells and cancer cells. Excess cohesion at telomeres can be induced by inhibition of tankyrase 1, a poly(ADP-ribose) polymerase that is required for resolution of telomere cohesion, or by overexpression of proteins required to establish telomere cohesion, the shelterin subunit TIN2 and the cohesin subunit SA1. Regardless of the method of induction, excess cohesion at telomeres in mitosis prevents a robust and efficient anaphase. SA1- or TIN2-induced excess cohesion and anaphase delay can be rescued by overexpression of tankyrase 1. Moreover, we show that primary fibroblasts, which accumulate excess telomere cohesion at mitosis naturally during replicative aging, undergo a similar delay in anaphase progression that can also be rescued by overexpression of tankyrase 1. Our study demonstrates that there are opposing forces that regulate telomere cohesion. The observation that cells respond to unresolved telomere cohesion by delaying (but not completely disrupting) anaphase progression suggests a mechanism for tolerating excess cohesion and maintaining telomere integrity. This attempt to deal with telomere damage may be ultimately futile for aging fibroblasts but useful for cancer cells.     

A Splicing Variant of NME1 Negatively Regulates NF-κB Signaling and Inhibits Cancer Metastasis by Interacting with IKKβ

IKKβ functions as a principal upstream activator of the canonical NF-κB pathway by phosphorylating IκB, leading to its proteasomal degradation. Because IKKβ is considered a therapeutic target, understanding its regulation may facilitate the design of efficient regulators of this molecule. Here, we report a novel IKKβ-interacting molecule, NME1L, a splicing variant of the NME1 protein. NME1 has attracted attention in cancer research because of its antimetastatic activity and reduced expression in multiple aggressive types of cancer. However, the effect was just moderate but not dramatic in anti-cancer activities. We found that only NME1L interacts with IKKβ. Exogenous expression of NME1L resulted in a potent decrease in TNFα-stimulated NF-κB activation, whereas knockdown of NME1/NME1L with shRNA enhanced activity of NF-κB. NME1L down-regulates IKKβ signaling by blocking IKKβ-mediated IκB degradation. When NME1L was introduced into highly metastatic HT1080 cells, the mobility was efficiently inhibited. Furthermore, in a metastasis assay, NME1L-expressing cells did not colonize the lung. Based on these results, NME1L is a potent antimetastatic protein and may be a useful weapon in the fight against cancers.     

Prevalence and Risk Factors of Clonorchiasis among Residents of Riverside Areas in Muju-gun,Jeollabuk-do,Korea

We evaluated the status of Clonorchis sinensis infection and potential risk factors among residents of riverside areas (Geumgang) in Muju-gun, Jeollabuk-do (Province), Korea. From January to February 2010, a total of 349 (171 males, 178 females) stool samples were collected and examined by the formalin-ether concentration technique. Also, village residents were interviewed using questionnaires to obtain information about C. sinensis infection-related risk factors. Overall egg-positive rate of C. sinensis was 13.2%. Egg-positive rates were significantly higher in males, farmers, and residents who had lived there more than 20 years, and in residents who had eaten raw freshwater fish than in opposite groups, respectively. However, there was no significant difference between age groups, education levels, cigarette smoking, alcohol drinking, health status, past history of infection, and experience of clonorchiasis medication and examination. Logistic regression analysis was performed to determine risk factors for clonorchiasis. On univariate analysis, the odds ratios for males, farmers, those who had lived there more than 20 years, and who had eaten raw freshwater fish were 2.41, 4.44, 3.16, and 4.88 times higher than those of the opposites, respectively. On multivariate analysis, the odds ratio of residents who had eaten raw freshwater fish was 3.2-fold higher than that of those who had not. These results indicate that residents living in Muju-gun, along the Geum River, Korea, have relatively high C. sinensis egg-positive rates, and the habit of eating raw freshwater fish was the major factor for the maintenance of clonorchiasis.     

Suppression of Cpn10 Increases Mitochondrial Fission and Dysfunction in Neuroblastoma Cells

To date, several regulatory proteins involved in mitochondrial dynamics have been identified. However, the precise mechanism coordinating these complex processes remains unclear. Mitochondrial chaperones regulate mitochondrial function and structure. Chaperonin 10 (Cpn10) interacts with heat shock protein 60 (HSP60) and functions as a co-chaperone. In this study, we found that down-regulation of Cpn10 highly promoted mitochondrial fragmentation in SK-N-MC and SH-SY5Y neuroblastoma cells. Both genetic and chemical inhibition of Drp1 suppressed the mitochondrial fragmentation induced by Cpn10 reduction. Reactive oxygen species (ROS) generation in 3-NP-treated cells was markedly enhanced by Cpn10 knock down. Depletion of Cpn10 synergistically increased cell death in response to 3-NP treatment. Furthermore, inhibition of Drp1 recovered Cpn10-mediated mitochondrial dysfunction in 3-NP-treated cells. Moreover, an ROS scavenger suppressed cell death mediated by Cpn10 knockdown in 3-NP-treated cells. Taken together, these results showed that down-regulation of Cpn10 increased mitochondrial fragmentation and potentiated 3-NP-mediated mitochondrial dysfunction in neuroblastoma cells.     

Renin inhibition activity by chitooligosaccharides

Six kinds of chitooligosaccharides (COSs) with different molecular weight (MW) and degree of deacetylation (DD) were prepared using ultrafiltration membrane reactor, and their renin inhibition modes were evaluated. All the COSs showed the renin-inhibitory activities with dose-dependent manner, and 90-COSs had the potent renin-inhibitory activity than that of 50-COSs. Among them, 90-MMWCOS (1000-5000Da) exhibits the highest activity with IC(50) value of 0.51mg/mL and acts as competitive inhibitor with K(i) value of 0.28mg/mL by Lineweaver-Burk and Dixon plots. These results indicated that DD value and MW of COSs are important factors affecting renin-inhibitory activity.     

Safety of Intracoronary Infusion of 20 Million C-Kit Positive Human Cardiac Stem Cells in Pigs

Background

There is mounting interest in using c-kit positive human cardiac stem cells (c-kit^pos hCSCs) to repair infarcted myocardium in patients with ischemic cardiomyopathy. A recent phase I clinical trial (SCIPIO) has shown that intracoronary infusion of 1 million hCSCs is safe. Higher doses of CSCs may provide superior reparative ability; however, it is unknown if doses >1 million cells are safe. To address this issue, we examined the effects of 20 million hCSCs in pigs.

Methods

Right atrial appendage samples were obtained from patients undergoing cardiac surgery. The tissue was processed by an established protocol with eventual immunomagnetic sorting to obtain in vitro expanded hCSCs. A cumulative dose of 20 million cells was given intracoronarily to pigs without stop flow. Safety was assessed by measurement of serial biomarkers (cardiac: troponin I and CK-MB, renal: creatinine and BUN, and hepatic: AST, ALT, and alkaline phosphatase) and echocardiography pre- and post-infusion. hCSC retention 30 days after infusion was quantified by PCR for human genomic DNA. All personnel were blinded as to group assignment.

Results

Compared with vehicle-treated controls (n=5), pigs that received 20 million hCSCs (n=9) showed no significant change in cardiac function or end organ damage (assessed by organ specific biomarkers) that could be attributed to hCSCs (P>0.05 in all cases). No hCSCs could be detected in left ventricular samples 30 days after infusion.

Conclusions

Intracoronary infusion of 20 million c-kit positive hCSCs in pigs (equivalent to ~40 million hCSCs in humans) does not cause acute cardiac injury, impairment of cardiac function, or liver and renal injury. These results have immediate translational value and lay the groundwork for using doses of CSCs >1 million in future clinical trials. Further studies are needed to ascertain whether administration of >1 million hCSCs is associated with greater efficacy in patients with ischemic cardiomyopathy.