Efficiency and Mechanism Evaluation of Magnolia officinalis Water Extract in Preventing Gastric Ulcer.

In this study we aimed at demonstrating the ability of Magnolia officinalis water extract to ameliorate gastric ulcers in in vitro and in vivo experiments. The gastric mucosa epithelial cell line, RGM 1, was pretreated with Magnolia officinalis water extract (0, 0.1, 1, 2, 5, or 10 mg/ml) and cultured in DMEM/F12 medium (pH 7.4) for 2 h and then in DMEM/F12 medium (pH 4.0) for 10 min. Magnolia officinalis water extract protected the cell viability and decreased reactive oxygen species formation by the acidic medium. In the in vivo experiment, Magnolia officinalis water extract (100 mg/kg) was administrated daily for 28 days in ICR mice via oral gavage, and then Shay's ulcer surgical method was performed to induce gastric ulcers. We analyzed the pH value of stomach acid and the pathological section, inflammation, and cannabinoid receptor type 2 (CB2) cDNA levels of the stomach. Magnolia officinalis water extract not only enhanced the pH value of stomach acid but also ameliorated the ulcer index and inflammation and increased CB2 expression effectively. These results suggest that Magnolia officinalis water extract might be used to decrease the incidence of gastric ulcer.

Nuclear envelope tethering inhibits the formation of ALT-associated PML bodies in ALT cells.

Telomere length homeostasis is essential for maintaining genomic stability and cancer proliferation. Telomerase-negative cancer cells undergo recombination-mediated alternative lengthening of telomeres. Telomeres associate with the nuclear envelope through the shelterin RAP1 and nuclear envelope SUN1 proteins. However, how the associations between telomeres and the nuclear envelope affect the progression of telomere recombination is not understood. Here, we show that telomere anchorage might inhibit telomere-telomere recombination. SUN1 depletion stimulates the formation of alternative lengthening of telomeres-associated promyelocytic leukemia bodies in ALT cells. In contrast, overexpression of a telomere-nuclear envelope-tethering chimera protein, RAP1-SUN1, suppresses APB formation. Moreover, inhibition of this nuclear envelope attachment alleviates the requirement of TOP3α for resolving the supercoiling pressure during telomere recombination. A coimmunoprecipitation assay revealed that the SUN1 N-terminal nucleoplasmic domain interacts with the RAP1 middle coil domain, and phosphorylation-mimetic mutations in RAP1 inhibit this interaction. However, abolishing the RAP1-SUN1 interaction does not hinder APB formation, which hints at the existence of another SUN1-dependent telomere anchorage pathway. In summary, our results reveal an inhibitory role of telomere-nuclear envelope association in telomere-telomere recombination and imply the presence of redundant pathways for the telomere-nuclear envelope association in ALT cells.

Selected ellipticine derivatives, known to target topoisomerase II, suppress the alternative lengthening of telomere (ALT) pathway in telomerase-negative cells.

BACKGROUND: DNA topoisomerase and telomerase enzymes are popular targets of several anti-tumor drugs. Smooth proceeding of telomeric recombination requires Topoisomerase II (Top2), which is involved in telomere-telomere recombination through functioning in relaxation of positive supercoils among the cells adopting telomerase-independent Alternative lengthening of telomere (ALT) pathway. Most of the inhibitors reported so far have been designed to targetsolely telomerase-positive cells, which can potentially lead to therapeutic failure because tumor cells treated with telomerase inhibitors can activate the ALT pathway for telomere maintenance. Knowing that ALT cells are more sensitive against a Top2 inhibitor, ICRF-93 agent, compared to telomerase-positive cells, we analyzed two selected ellipticine derivatives that we recently reported as TopII-targeting compounds, to assess their effects on the formation of DNA breaks and suppression of ALT pathway. METHODS: Cell viability, Comet, C-Circle assays, dot blot, immunofluorescence staining, and telomere fluorescence in situ hybridization (FISH) staining were used for determining the effect of the compounds on ALT status of tumor cells. RESULTS AND CONCLUSIONS: Treatment of ALT cells with ellipticine derivatives resulted in the formation of DNA breaks and suppression of ALT-associated phenotypes in vitro. Our results will contribute to the development of therapeutic strategies combining telomerase and ALT pathway inhibitors.

Probing Charge Transport Difference in Parallel and Vertical Layered Electronics with Thin Graphite Source/Drain Contacts.

In the present study, we aim to help improve the design of van der Waals stacking, i.e., vertical 2D electronics, by probing charge transport differences in both parallel and vertical conducting channels of layered molybdenum disulfide (MoS2), with thin graphite acting as source and drain electrodes. To avoid systematic errors and variable contact contributions to the MoS2 channel, parallel and vertical electronics are all fabricated and measured on the same conducting material. Large differences in the on/off current ratio, mobility, and charge fluctuations, between parallel and vertical electronics are evident in electrical performance as well as in charge transport mechanisms. Further insights are drawn from a well-constrained analysis of both temperature-dependent current-voltage characteristics and low-frequency (LF) current fluctuations. This work offers significant insight into the fundamental understanding of charge transport and the development of future layered-materials-based integration technology.

PARP1 controls KLF4-mediated telomerase expression in stem cells and cancer cells.

Telomerase is highly expressed in cancer and embryonic stem cells (ESCs) and implicated in controlling genome integrity, cancer formation and stemness. Previous studies identified that Krüppel-like transcription factor 4 (KLF4) activates telomerase reverse transcriptase (TERT) expression and contributes to the maintenance of self-renewal in ESCs. However, little is known about how KLF4 regulates TERT expression. Here, we discover poly(ADP-ribose) polymerase 1 (PARP1) as a novel KLF4-interacting partner. Knockdown of PARP1 reduces TERT expression and telomerase activity not only in cancer cells, but also in human and mouse ESCs. Recruitment of KLF4 to TERT promoter is reduced in PARP1-suppressed cells. The poly(ADP-ribose) polymerase activity is dispensable, while the oligo(ADP-ribose) polymerase activity is required for the PARP1- and KLF4-mediated TERT activation. Repression of Parp1 in mouse ESCs decreases expression of pluripotent markers and induces differentiation. These results suggest that PARP1 recruits KLF4 to activate telomerase expression and stem cell pluripotency, indicating a positive regulatory role of the PARP1-KLF4 complex in telomerase expression in cancer and stem cells.

Yeast Cip1 is activated by environmental stress to inhibit Cdk1-G1 cyclins via Mcm1 and Msn2/4.

Upon environmental changes, proliferating cells delay cell cycle to prevent further damage accumulation. Yeast Cip1 is a Cdk1 and Cln2-associated protein. However, the function and regulation of Cip1 are still poorly understood. Here we report that Cip1 expression is co-regulated by the cell-cycle-mediated factor Mcm1 and the stress-mediated factors Msn2/4. Overexpression of Cip1 arrests cell cycle through inhibition of Cdk1-G1 cyclin complexes at G1 stage and the stress-activated protein kinase-dependent Cip1 T65, T69, and T73 phosphorylation may strengthen the Cip1and Cdk1-G1 cyclin interaction. Cip1 accumulation mainly targets Cdk1-Cln3 complex to prevent Whi5 phosphorylation and inhibit early G1 progression. Under osmotic stress, Cip1 expression triggers transient G1 delay which plays a functionally redundant role with another hyperosmolar activated CKI, Sic1. These findings indicate that Cip1 functions similarly to mammalian p21 as a stress-induced CDK inhibitor to decelerate cell cycle through G1 cyclins to cope with environmental stresses.A G1 cell cycle regulatory kinase Cip1 has been identified in budding yeast but how this is regulated is unclear. Here the authors identify cell cycle (Mcm1) and stress-mediated (Msn 2/4) transcription factors as regulating Cip1, causing stress induced CDK inhibition and delay in cell cycle progression.

Genistein suppresses the proliferation of telomerase-negative cells.

In both tumor and yeast cells that lack telomerase, telomeres are maintained via an alternative recombination mechanism. In this study, we tested genistein, a potential TOP2 inhibitor required for telomere-telomere recombination, on the repression of telomere-telomere recombination. Genistein on the repression of type II recombination on a tlc1 yeast strain was examined by the telomeric DNA structures using Southern blot analysis. Telomere patterns of freshly dissected tlc1 spores containing an empty plasmid (pYES2) or a yeast TOP2 (yTOP2) plasmid were analyzed. The results indicated that the reintroduction of TOP2 recovered the type II pattern, implying genistein in the blockage of type II survivors in the tlc1 strain. The effects of genistein on both tlc1 and tlc1 rad 51 strains in liquid and solid mediums were also examined. Finally, treatment of 10 µmol/L of genistein showed inhibitory effect on the growth of telomerase-negative U2OS alternative lengthening of telomere (ALT) cells, but not in telomerase-positive HCT116 cells. These results provide evidences that the inhibitory effects of genistein on telomerase-negative cells depend on type II recombination pathway in yeast and the ALT pathway in human tumors.

Topoisomerase II inhibition suppresses the proliferation of telomerase-negative cancers.

Telomere maintenance is required for chromosome stability, and telomeres are typically elongated by telomerase following DNA replication. In both tumor and yeast cells that lack telomerase, telomeres are maintained via an alternative recombination mechanism. Previous studies have indicated that yeast Sgs1 and Top3 may work together to remove highly negative supercoils that are generated from recombination. However, the mechanism by which cells eradicate highly positive supercoils during recombination remains unclear. In the present study, we demonstrate that TOP2 is involved in telomere-telomere recombination. Disturbance of telomeric structure by RIF1 or RIF2 deletion alleviates the requirement for TOP2 in telomere-telomere recombination. In human telomerase-negative alternative lengthening of telomere (ALT) cells, TOP2α or TOP2ß knockdown decreases ALT-associated PML bodies, increases telomere dysfunction-induced foci and triggers telomere shortening. Similar results were observed when ALT cells were treated with ICRF-193, a TOP2 inhibitor. Importantly, ICRF-193 treatment blocks ALT-associated phenotypes in vitro, causes telomere shortening, and inhibits ALT cell proliferation in mice. Taken together, these findings imply that TOP2 is involved in the ALT pathway, perhaps by resolving the highly positive supercoil structure at the front of the helicase. Inhibition of topoisomerase II may be a promising therapeutic approach that can be used to prevent cell proliferation in ALT-type cancer cells.

Instability of succinate dehydrogenase in SDHD polymorphism connects reactive oxygen species production to nuclear and mitochondrial genomic mutations in yeast.

AIMS: Mitochondrial succinate dehydrogenase (SDH) is an essential complex of the electron transport chain and tricarboxylic acid cycle. Mutations in the human SDH subunit D frequently lead to paraganglioma (PGL), but the mechanistic consequences of the majority of SDHD polymorphisms have yet to be unraveled. In addition to the originally discovered yeast SDHD subunit Sdh4, a conserved homolog, Shh4, has recently been identified in budding yeast. To assess the pathogenic significance of SDHD mutations in PGL patients, we performed functional studies in yeast. RESULTS: SDHD protein expression was reduced in SDHD-related carotid body tumor tissues. A BLAST search of SDHD to the yeast protein database revealed a novel protein, Shh4, that may have a function similar to human SDHD and yeast Sdh4. The missense SDHD mutations identified in PGL patients were created in Sdh4 and Shh4, and, surprisingly, a severe respiratory incompetence and reduced expression of the mutant protein was observed in the sdh4Δ strain expressing shh4. Although shh4Δ cells showed no respiratory-deficient phenotypes, deletion of SHH4 in sdh4Δ cells further abolished mitochondrial function. Remarkably, sdh4Δ shh4Δ strains exhibited increased reactive oxygen species (ROS) production, nuclear DNA instability, mtDNA mutability, and decreased chronological lifespan. INNOVATION AND CONCLUSION: SDHD mutations are associated with protein and nuclear and mitochondrial genomic instability and increase ROS production in our yeast model. These findings reinforce our understanding of the mechanisms underlying PGL tumorigenesis and point to the yeast Shh4 as a good model to investigate the possible pathogenic relevance of SDHD in PGL polymorphisms.

Analysis of the cellulose synthase genes associated with primary cell wall synthesis in Bambusa oldhamii.

The synthesis of cell wall polysaccharides is highly active in rapidly growing bamboo shoots. We cloned a set of BoCesA cDNAs that encode cellulose synthase from bamboo (Bambusa oldhamii) and investigated the expression patterns of the BoCesA2, BoCesA5, BoCesA6 and BoCesA7 genes. The four BoCesA genes were differentially expressed in the different parts of growing bamboo shoots, in various organs, and in multiple shoots that were cultured in vitro. They were down-regulated by alpha-naphthaleneacetic acid and differentially affected by thidiazuron in the multiple shoots. In situ RT-PCR analyses demonstrated that BoCesA2, BoCesA5, BoCesA6, and BoCesA7 mRNAs were present throughout the base and the internode regions of the etiolated shoots that emerged from pseudorhizomes, and in the internode regions of the juvenile branch shoots that emerged from nodes of mature bamboo culms; however, the expression of the four genes in the lignified internode of the branch shoot was predominantly detected in the center of the vascular bundles. Our results for cDNA cloning, expression analyses, and phylogenetic analysis suggest that the 10 BoCesA genes cloned from the etiolated bamboo shoots participate in cellulose synthesis in the primary cell walls of the growing bamboo, and that at least three additional BoCesA genes involved in cellulose synthesis in the secondary walls may be present in the bamboo genome. The expressions of BoCesA genes may be under fine control in response to the various developmental stages and physiological conditions of bamboo.

Molecular characterization and expression of four cDNAs encoding sucrose synthase from green bamboo Bambusa oldhamii.

Bamboo is distinguished by its rapid growth. To investigate sucrose metabolism in this plant, we cloned the cDNAs encoding sucrose synthase (SuS) from Bambusa oldhamii and investigated their expression in growing shoots and leaves. Four cDNA clones, BoSus1, BoSus2, BoSus3 and BoSus4, were isolated by screening a cDNA library from etiolated bamboo shoots. Recombinant BoSuS proteins were produced in Escherichia coli and purified by immobilized metal affinity chromatography and ultrafiltration. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine the abundance of the transcript of each gene. BoSus1 and BoSus3 may be duplicate or homeologous genes, the sequences of which show high identity. Similarly, BoSus2 shows high identity with BoSus4. Kinetic analysis showed that the two BoSuS isoforms of each type had similar michaelis constant (Km) values for sucrose, but different values for UDP. The four genes were expressed in various bamboo organs but were differentially regulated. The increase in the abundance of their mRNA paralleled the growth rate of the bamboo. The results suggest that, in bamboo, SuS is encoded by at least four genes, each with a specific role in providing substrates for the polysaccharide biosynthesis and/or energy production necessary to support the rapid growth of this species.