Changes of Hydrolytic Activities of Buckwheat α-Glucosidase by Acetylation with N-Acetylimidazole

A treatment of buckwheat α-glucosidase with N-acetylimidazole brought about the acétylation of 6.4 tyrosyl residues, 0.38 free sulfhydryl groups and about 50% of free amino groups, and the decrease in the hydrolytic activities toward maltooligosaccharides (G₂~G₈, G13) and soluble starch. The affinities for the substrate other than maltose were diminished by the modification and the extent of the reduction of the affinities was apparently dependent on the degree of polymerization of maltooligosaccharides, while the affinity for phenyl α- maltoside was increased. The treatment of the acetylated enzyme with hydroxylamine resulted in the complete restration of the affinities for all substrates tested. It seems that these facts were due to the acétylation of several tyrosyl residues located in or near certain subsites of the enzyme. About 25 % of the hydrolytic activity remained inert in spite of the deacetylation with hydroxylamine, which was assumed to be attributed to the partial modification of free sulfhydryl group localized closely near the catalytic site of the enzyme.     

Formation of Rhamnolipid by Pseudomonas aeruginosa and its Function in Hydrocarbon Fermentation

Screening test for obtaining growth stimulant (GS) produced by a hydrocarbon-utilizing bacterium, Pseudomonas aeruginosa S₇B₁, was carried out. In consequence, the anthrone positive substance was most effective on the growth of this strain. Although the growth of this strain on glucose medium had no relation with the addition of GS, the growth on n-hexadecane medium was remarkably stimulated by the addition of GS. This effect of GS seemed to be specific on the growth of P. aeruginosa. GS which had a strong surface activity and emulsifying power was comfirmed to be rhamnolipid.     

Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.     

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.     

Enhanced sampling simulations to construct free-energy landscape of protein–partner substrate interaction

Molecular dynamics (MD) simulations using all-atom and explicit solvent models provide valuable information on the detailed behavior of protein–partner substrate binding at the atomic level. As the power of computational resources increase, MD simulations are being used more widely and easily. However, it is still difficult to investigate the thermodynamic properties of protein–partner substrate binding and protein folding with conventional MD simulations. Enhanced sampling methods have been developed to sample conformations that reflect equilibrium conditions in a more efficient manner than conventional MD simulations, thereby allowing the construction of accurate free-energy landscapes. In this review, we discuss these enhanced sampling methods using a series of case-by-case examples. In particular, we review enhanced sampling methods conforming to trivial trajectory parallelization, virtual-system coupled multicanonical MD, and adaptive lambda square dynamics. These methods have been recently developed based on the existing method of multicanonical MD simulation. Their applications are reviewed with an emphasis on describing their practical implementation. In our concluding remarks we explore extensions of the enhanced sampling methods that may allow for even more efficient sampling.     

NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

Recent studies have shown that Notch signaling is involved in many types of cancers, including oral squamous cell carcinomas (OSCCs). However, the role of Notch signaling in the tumor microenvironment is not yet fully understood. In this study, we investigated the roles of NOTCH3 signaling in cancer associated fibroblasts (CAFs) in OSCCs. Immunohistochemical study of 93 human tongue OSCC cases indicated that about one third of OSCCs showed NOTCH3 expression in CAFs, and that this expression significantly correlated with tumor-size. In vitro study showed that OSCC cell lines, especially HO1-N-1 cells stimulated NOTCH3 expression in normal human dermal fibroblasts (NHDFs) through direct cell-to-cell contact. Immunohistochemical and morphometric analysis using human OSCC samples demonstrated that NOTCH3 expression in CAFs significantly correlated with micro-vessel density in cancer stroma. In vitro angiogenesis assays involving co-culture of NHDFs with HO1-N-1 and human umbilical endothelial cells (HUVECs), and NOTCH3 knockdown in NHDFs using siRNA, demonstrated that HO1-N-1 cells significantly promoted tube formation dependent on NOTCH3-expression in NHDFs. Moreover, NOTCH3 expression in CAFs was related to poor prognosis of the OSCC patients. This work provides a new insight into the role of Notch signaling in CAFs associated with tumor angiogenesis and the possibility of NOTCH3-targeted molecular therapy in OSCCs.     

Meltrin beta expressed in cardiac neural crest cells is required for ventricular septum formation of the heart

The heart is divided into four chambers by membranous septa and valves. Although evidence suggests that formation of the membranous septa requires migration of neural crest cells into the developing heart, the functional significance of these neural crest cells in the development of the endocardial cushion, an embryonic tissue that gives rise to the membranous appendages, is largely unknown. Mice defective in the protease region of Meltrin beta/ADAM19 show ventricular septal defects and defects in valve formation. In this study, by expressing Meltrin beta in either endothelial or neural crest cell lineages, we showed that Meltrin beta expressed in neural crest cells but not in endothelial cells was required for formation of the ventricular septum and valves. Although Meltrin beta-deficient neural crest cells migrated into the heart normally, they could not properly fuse the right and left ridges of the cushion tissues in the proximal outflow tract (OT), and this led to defects in the assembly of the OT and AV cushions forming the ventricular septum. These results genetically demonstrated a critical role of cardiac neural crest cells expressing Meltrin beta in triggering fusion of the proximal OT cushions and in formation of the ventricular septum.     

A novel in vitro co-culture model to examine contact formation between astrocytic processes and cerebral vessels

Here, we developed a novel in vitro co-culture model, in which process-bearing astrocytes and isolated cerebral microvessels from mice were co-cultured. Astrocytes formed contacts with microvessels from both adult and neonatal mice. However, concentrated localization of the immunofluorescence signal for aquaporin-4 (AQP4) at contact sites between perivascular endfoot processes and blood vessels was only detected with neonatal mouse microvessels. Contact between astrocytic processes and microvessels was retained, whereas concentrated localization of AQP4 signal at contact sites was lost, by knockdown of dystroglycan or α-syntrophin, reflecting polarized localization of AQP4 at perivascular regions in the brain. Further, using our in vitro co-culture model, we found that astrocytes predominantly extend processes to pericytes located at the abluminal surface of microvessels, providing additional evidence that this model is representative of the in vivo situation. Altogether, we have developed a novel in vitro co-culture model that can reproduce aspects of the in vivo situation and is useful for assessing contact formation between astrocytes and blood vessels.