Isolation and Characterization of Oligosaccharides from the Hydrolyzate of Larch Wood Glucomannan with Endo-β-d-Mannanase

The glucomannan isolated from larch holocellulose was hydrolyzed by a purified endo-d-β-mannanase. The products were fractionated by gel filtration on a Polyacrylamide gel in water and partition chromatography on ion exchange resins in 80% ethanol. The following oligosaccharides were isolated and identified: (a) 4-O-β-d-Manp-d-Man, (b) 4-O-β-d-Glcp-d-Man, (c) 4-O-β-d-Glcp-d-Glc, (d) O-β-d-Manp-(1 →4)-O-β-d-Manp-(1 →4)-d-Man, (e) O-β-dGlcp-(l →4)-O-β-d-Manp-(l →4)-d-Man, (f) O-β-d-Manp-(l →4)-Oβ-d-Glcp-(l →4)-d-Man, (g) O-β-d-Manp-(l →4)-O-[α-d-Galp-(l →6)]-d-Man, (h) O-β-d-Manp-(l →4)-O-β-d-Manp-(l →4)-O-β-d-Manp-(l →4)-d-Man, and (i) O-β-d-Glcp-(1 →4)-O-β-d-Manp-(1 →4)-O-β-d-Manp-(1 →4)-d-Man.     

Reassignment of a rare sense codon to a non-canonical amino acid in Escherichia coli

The immutability of the genetic code has been challenged with the successful reassignment of the UAG stop codon to non-natural amino acids in Escherichia coli. In the present study, we demonstrated the in vivo reassignment of the AGG sense codon from arginine to l-homoarginine. As the first step, we engineered a novel variant of the archaeal pyrrolysyl-tRNA synthetase (PylRS) able to recognize l-homoarginine and l-N⁶-(1-iminoethyl)lysine (l-NIL). When this PylRS variant or HarRS was expressed in E. coli, together with the AGG-reading tRNA^Pyl_CCU molecule, these arginine analogs were efficiently incorporated into proteins in response to AGG. Next, some or all of the AGG codons in the essential genes were eliminated by their synonymous replacements with other arginine codons, whereas the majority of the AGG codons remained in the genome. The bacterial host''s ability to translate AGG into arginine was then restricted in a temperature-dependent manner. The temperature sensitivity caused by this restriction was rescued by the translation of AGG to l-homoarginine or l-NIL. The assignment of AGG to l-homoarginine in the cells was confirmed by mass spectrometric analyses. The results showed the feasibility of breaking the degeneracy of sense codons to enhance the amino-acid diversity in the genetic code.     

Vasorelaxant effects of macrocyclic bis(bibenzyls) from liverworts

Vasorelaxant effects of a series of bis(bibenzyls) from liverworts such as Marchantia polymorpha and Marchantia paleacea on rat aorta demonstrated that they relaxed phenylephrine (PE)-induced contractions, which may be mediated through the increased release of NO from endothelial cells as well as opening of K(+) channels, and inhibition of Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCs) and/or receptor-operated Ca(2+) channels (ROCs). Structure-activity relationship based on their structures was discussed. The presence of two aromatic rings which can be connected through two atoms bridge spacer may play an important role for vasorelaxant effect.     

Differential subcellular localization of ACTH and α-MSH in corticotropes of the rat anterior pituitary

Summary Specific antisera to -melanotropin (-MSH) and corticotropin (ACTH 1-39) were used to obtain immunocytochemical evidence for the differential localization of -MSH and ACTH in the secretory granules of corticotropes of rat anterior pituitary. The specificity of the antisera was established by binding ¹³¹I-labeled -MSH and ACTH 1-39 to their respective antisera. Double-labeling immunocytochemistry (for -MSH, ferritin; for ACTH, colloidal gold) was performed. Some secretory granules were labeled with ferritin particles (-MSH), whereas others contained gold particles (ACTH). Only a few granules showed both ACTH and -MSH. In typical corticotropes (stellate in form with a small number of secretory granules aligned along the cell periphery) only some of the secretory granules that were labeled with anti-ACTH serum were also immunoreactive to anti--MSH. In atypical corticotropes (polygonal in shape and containing a large number of secretory granules) almost all of the immunoreactive ACTH secretory granules were also positive to anti--MSH serum. An intermediate type of corticotrope was observed containing a small number of secretory granules, almost all of which were labeled with anti--MSH. Thus, rat anterior pituitary corticotropes may be classified into three types according to the distribution and content of -MSH. The light-microscopic immuncytochemistry provided similar results.     

Design, synthesis, and biological evaluation of 4-phenylpyrrole derivatives as novel androgen receptor antagonists

A series of 4-phenylpyrrole derivatives D were designed, synthesized, and evaluated for their potential as novel orally available androgen receptor antagonists therapeutically effective against castration-resistant prostate cancers. 4-Phenylpyrrole compound 1 exhibited androgen receptor (AR) antagonistic activity against T877A and W741C mutant-type ARs as well as wild-type AR. An arylmethyl group incorporated into compound 1 contributed to enhancement of antagonistic activity. Compound 4n, 1-{[6-chloro-5-(hydroxymethyl)pyridin-3-yl]methyl}-4-(4-cyanophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonitrile exhibited inhibitory effects on tumor cell growth against the bicalutamide-resistant LNCaP-cxD2 cell line as well as the androgen receptor-dependent JDCaP cell line in a mouse xenograft model. These results demonstrate that this series of pyrrole compounds are novel androgen receptor antagonists with efficacy against prostate cancer cells, including castration-resistant prostate cancers such as bicalutamide-resistant prostate cancer.     

Sunitinib induces apoptosis in pheochromocytoma tumor cells by inhibiting VEGFR2/Akt/mTOR/S6K1 pathways through modulation of Bcl-2 and BAD

Sunitinib is an oral multitargeted receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity that mainly targets vascular endothelial growth factor receptors (VEGFRs). Very recently, sunitinib has been shown to be an active agent for the treatment of malignant pheochromocytomas. However, it is unclear whether sunitinib acts only through an antiangiogenic mechanism or whether it may also directly target tumor cells. Sunitinib markedly induced apoptosis of PC12 cells in a dose-dependent and time-dependent manner. Furthermore, in support of these findings, we found that sunitinib induced a reduction in the expression of the antiapoptotic molecule Bcl-2 as well as dephosphorylation of the proapoptotic molecule BAD, which results in the activation of BAD in these cells. Consistent with these apoptotic effects, our results showed that sunitinib inhibited phosphorylation of Akt and mTOR and was followed by a reduction of S6K1, which is a well-known target of mTOR. Knockdown of VEGFR-2 attenuated the sunitinib-induced effects, including apoptosis and inhibition of signaling pathways such as the phosphorylation of Akt as well as mTOR, and Bcl-2, which confirmed that these effects could be mediated by VEGFR-2. In addition, silencing of S6K1 induced apoptosis accompanied by a decrease in the phosphorylation of BAD and Bcl-2, similar to that observed with sunitinib treatment. Thus, these results together suggest that sunitinib initially exerts its apoptotic effect through the inhibition of VEGFR-2, which, when followed by reduction of its downstream effectors, including Akt/mTOR/S6K1, may lead to inhibition of the antiapoptotic molecule Bcl-2 and activation of the proapoptotic molecule BAD in PC12 cells. However, PC12 cells do not precisely reflect the pathogenesis of malignant cells. Therefore, we confirmed the key findings by replicating these experiments in human neuroblastoma SK-N-SH cells.     

Laser-induced Propagation and Destruction of Amyloid β Fibrils

The amyloid deposition of amyloid β (Aβ) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of β₂-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on Aβ fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed Aβ fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.