Direct In Vivo Reprogramming with Sendai Virus Vectors Improves Cardiac Function after Myocardial Infarction

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Synthesis of phenserine analogues and evaluation of their cholinesterase inhibitory activities

Phenserine is a potentially attractive drug for Alzheimer's disease. In order to further expand SAR study for inhibitions of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), the methyl group at the 3a-position of phenserine was replaced with an alkyl or alkenyl group, and its phenylcarbamoyl moiety was substituted at the o- or p-position. The synthetic methodology for these phenserine analogues includes the efficient cascade reactions for introduction of the 3a-substituent and assembly of the quaternary carbon center followed by reductive cyclization to the key pyrroloindoline structure. The bulkiness of the substituent at 3a-position of phenserine derivatives tends to reduce the inhibitory effect on AChE activity in the following order: methyl>ethyl>vinyl>propyl≈allyl>reverse-prenyl groups. Among the series synthesized, the 3a-ethyl derivative demonstrated the highest AChE selectivity. In construct, the 3a-reverse-prenyl derivative indicated modest BuChE selectivity.     

Aberrant Glycogen Synthase Kinase 3β Is Involved in Pancreatic Cancer Cell Invasion and Resistance to Therapy

Background and Purpose

The major obstacles to treatment of pancreatic cancer are the highly invasive capacity and resistance to chemo- and radiotherapy. Glycogen synthase kinase 3β (GSK3β) regulates multiple cellular pathways and is implicated in various diseases including cancer. Here we investigate a pathological role for GSK3β in the invasive and treatment resistant phenotype of pancreatic cancer.

Methods

Pancreatic cancer cells were examined for GSK3β expression, phosphorylation and activity using Western blotting and in vitro kinase assay. The effects of GSK3β inhibition on cancer cell survival, proliferation, invasive ability and susceptibility to gemcitabine and radiation were examined following treatment with a pharmacological inhibitor or by RNA interference. Effects of GSK3β inhibition on cancer cell xenografts were also examined.

Results

Pancreatic cancer cells showed higher expression and activity of GSK3β than non-neoplastic cells, which were associated with changes in its differential phosphorylation. Inhibition of GSK3β significantly reduced the proliferation and survival of cancer cells, sensitized them to gemcitabine and ionizing radiation, and attenuated their migration and invasion. These effects were associated with decreases in cyclin D1 expression and Rb phosphorylation. Inhibition of GSK3β also altered the subcellular localization of Rac1 and F-actin and the cellular microarchitecture, including lamellipodia. Coincident with these changes were the reduced secretion of matrix metalloproteinase-2 (MMP-2) and decreased phosphorylation of focal adhesion kinase (FAK). The effects of GSK3β inhibition on tumor invasion, susceptibility to gemcitabine, MMP-2 expression and FAK phosphorylation were observed in tumor xenografts.

Conclusion

The targeting of GSK3β represents an effective strategy to overcome the dual challenges of invasiveness and treatment resistance in pancreatic cancer.     

Demonstration of the hepatocyte growth factor signaling pathway in the in vitro neuritogenic activity of chondroitin sulfate from ray fish cartilage

Background

Chondroitin sulfate (CS) is a ubiquitous component of the cell surface and extracellular matrix and its sugar backbone consists of repeating disaccharide units: D-glucuronic acid (GlcUA)β1-3N-acetyl-D-galactosamine (GalNAc). Although CS participates in diverse biological processes such as growth factor signaling and the nervous system's development, the mechanism underlying the functions is not well understood.

Methods

CS was isolated from ray fish cartilage, an industrial waste, and its structure and neurite outgrowth-promoting (NOP) activity were analyzed to investigate a potential application to nerve regeneration.

Results

The major disaccharide unit in the CS preparation was GlcUA-GalNAc(6-O-sulfate) (61.9%). Minor proportions of GlcUA-GalNAc(4-O-sulfate) (27.0%), GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate) (8.5%), and GlcUA-GalNAc (2.7%) were also detected. The preparation showed NOP activity in vitro, and this activity was suppressed by antibodies against hepatocyte growth factor (HGF) and its receptor c-Met, suggesting the involvement of the HGF signaling pathway in the expression of the in vitro NOP activity of the CS preparation. The specific binding of HGF to the CS preparation was also demonstrated by surface plasmon resonance spectroscopy.

Conclusions and general significance

The NOP activity of CS from ray cartilage was demonstrated to be expressed through the HGF signaling pathway, suggesting that ray cartilage CS may be useful for studying the cooperative function of CS and HGF.     

PLRP2 selectively localizes synaptic membrane proteins via acyl-chain remodeling of phospholipids

The plasma membrane of neurons consists of distinct domains, each of which carries specialized functions and a characteristic set of membrane proteins. While this compartmentalized membrane organization is essential for neuronal functions, it remains controversial how neurons establish these domains on the laterally fluid membrane. Here, using immunostaining, lipid-MS analysis and gene ablation with the CRISPR/Cas9 system, we report that the pancreatic lipase-related protein 2 (PLRP2), a phospholipase A1 (PLA1), is a key organizer of membrane protein localization at the neurite tips of PC12 cells. PLRP2 produced local distribution of 1-oleoyl-2-palmitoyl-PC at these sites through acyl-chain remodeling of membrane phospholipids. The resulting lipid domain assembled the syntaxin 4 (Stx4) protein within itself by selectively interacting with the transmembrane domain of Stx4. The localized Stx4, in turn, facilitated the fusion of transport vesicles that contained the dopamine transporter with the domain of the plasma membrane, which led to the localized distribution of the transporter to that domain. These results revealed the pivotal roles of PLA1, specifically PLRP2, in the formation of functional domains in the plasma membrane of neurons. In addition, our results suggest a mode of membrane organization in which the local acyl-chain remodeling of membrane phospholipids controls the selective localization of membrane proteins by regulating both lipid-protein interactions and the fusion of transport vesicles to the lipid domain.     

Analysis of population structure among Korean and Japanese Legionella pneumophila isolates using hsp60 sequences

The population structure of Korean (150 strains) and Japanese (92 strains) Legionella pneumophila isolates along with 18 reference strains were investigated using hsp60 sequence (1647 bp) analysis. Twelve clonal subgroups (hsP-I to hsP-X and hsF-I and hsF-II) were designated on the hsp60 tree, inferred from representative sequences using the neighbor-joining method. Some of the isolates showed unique subgroups depending on the source of isolates, including hsP-I, hsF-I, and hsF-II from cooling tower water, and subgroups hsP-VIII and hsP-X from circulating hot water bath. These subgroups may be useful for epidemiological studies to chase or specify sources of infection in Korea and Japan.     

C. elegans pharyngeal morphogenesis requires both de novo synthesis of pyrimidines and synthesis of heparan sulfate proteoglycans

The C. elegans pharynx undergoes elongation and morphogenesis to its characteristic bi-lobed shape between the 2- and 3-fold stages of embryogenesis. During this period, the pharyngeal muscles and marginal cells forming the isthmus between the anterior and posterior pharyngeal bulbs elongate and narrow. We have identified the spontaneous mutant pyr-1(cu8) exhibiting defective pharyngeal isthmus elongation, cytoskeletal organization defects, and maternal effect lethality. pyr-1 encodes CAD, a trifunctional enzyme required for de novo pyrimidine synthesis, and pyr-1(cu8) mutants are rescued by supplying exogenous pyrimidines. Similar pharyngeal defects and maternal effect lethality were found in sqv-1, sqv-8, rib-1 and rib-2 mutants, which affect enzymes involved in heparan sulfate proteoglycan (HSPG) synthesis. rib-1 mutant lethality was enhanced in a pyr-1 mutant background, indicating that HSPG synthesis is very sensitive to decreased pyrimidine pools, and HS disaccharides are moderately decreased in both rib-1 and pyr-1 mutants. We hypothesize that HSPGs are necessary for pharyngeal isthmus elongation, and pyr-1 functions upstream of proteoglycan synthesizing enzymes by providing precursors of UDP-sugars essential for HSPG synthesis.     

HuC:Kaede, a useful tool to label neural morphologies in networks in vivo

A simple and efficient procedure for labeling neurons is a prerequisite for investigating the development of neural networks in zebrafish. To label neurons we used Kaede, a fluorescent protein with a photoconversion property allowing conversion from green to red fluorescence following irradiation with UV or violet light. We established a zebrafish stable transgenic line, Tg(HuC:Kaede), expressing Kaede in neurons under the control of the HuC promoter. This transgenic line was used to label a small number of neurons in the trigeminal ganglion. Also, using embryos injected with the transgene, we labeled peripheral axon arbors of a Rohon-Beard neuron at 4 days postfertilization and observed the dendrite development of a tectal neuron for 3 days. These data indicate that Kaede is a useful tool to selectively label neural networks in zebrafish.     

Generation of reactive oxygen species undergoing redox cycle of nostocine A: a cytotoxic violet pigment produced by freshwater cyanobacterium Nostoc spongiaeforme

Nostocine A (1) is an extracellular cytotoxic violet pigment produced by the freshwater cyanobacterium, Nostoc spongiaeforme TISTR 8169. Treatment with 1 was found to accelerate the generation of reactive oxygen species (ROS) in the green alga, Chlamydomonas reinhardtii, in the light. In vitro analysis revealed that 1 specifically eliminated superoxide radical anion (O(2)(-)) among several ROS tested. During the course of the reaction, oxygen (O(2)) was simultaneously synthesized and the O(2) synthesizing rate increased with the amount of 1 added. In contrast, O(2)(-) generation occurred when NADPH or NADH was added to a solution of 1 under aerobic condition. The reduction potential of 1 is very similar to that of O(2) indicating that 1 and O(2) can easily exchange electrons depending on the mass balance between their oxidized and reduced forms. Based on these results, the following hypothesis is formulated for the mechanism of intracellular ROS generation by treatment with 1: 1 taken into the target cells is reduced specifically by intracellular reductants such as NAD(P)H. When the O(2) level is sufficiently higher than that of 1, the reduced product of 1 is immediately oxidized by O(2). This is accompanied by the synthesis of O(2)(-) from O(2). The generation of O(2)(-) successively occurs, undergoing repeated redox cycles of 1, when the levels of the reductant and O(2) are still dominant to promote these reactions. This similar intracellular ROS generation mechanism to that of paraquat may cause the cytotoxicity.