Cytoplasmic filaments and cellular wound healing in amoeba proteus

The flexibility and self-healing properties of animal cell surface membranes are well known. These properties have been best exploited in various micrurgical studies on living cells (2, 3), especially in amoebae (7, 20). During nuclear transplantation in amoebae, the hole in the membrane through which a nucleus passes can have a diameter of 20-30 μm, and yet such holes are quickly sealed, although some cytoplasm usually escapes during the transfer. While enucleating amoebae in previous studies, we found that if a very small portion of a nucleus was pushed through the membrane and exposed to the external medium, the amoeba expelled such a nucleus on its own accord. When this happened, a new membrane appeared to form around the embedded portion of the nucleus and no visible loss of cytoplasm occurred during nuclear extrusion. In the present study, we examined amoebae that were at different stages of expelling partially exposed nuclei, to follow the sequence of events during the apparent new membrane formation. Unexpectedly, we found that a new membrane is not formed around the nucleus from inside but a hole is sealed primarily by a constriction of the existing membrane, and that cytoplasmic filaments are responsible for the prevention of the loss of cytoplasm.     

Synthesis of 2,2′-Anhydro-2-Hydroxy- and 6,2′-Anhydro-6-Hydroxy-1-β-D-Arabindfuranosylnicotinamide as Conformationally Restricted Nicotinamide Nucleoside Analogs1

Abstract

1-(β-D-Ribofuranosy1)-2(1H)-pyridone-3-carboxamide (6a) and the 6(1H)-pyridone derivative (6b) were prepared by condensation of 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose (3) with 2- and 6-hydroxynicotinic acid, respectively, to 4a and 4b, followed by conversion of the carboxylic acid function of 4a,b into their corresponding carboxamides 5, and then deprotection of 5. Bath 6a and 6b were then treated with 1,3-dichlom-1,1,3,3-tetraisopropyldisiloxane to give the corresponding 3′,5′-O-TPDS derivatives, 7a and 7b. Mesylation of 7a,b with mesyl chloride in pyridine afforded the stable, protected mesylates 8a,b. Upon de-O-silylation of 8a,b with ET₃NHF gave a mixture of unprotectd mesylates 9a,b and 2,2-anhydro- and 6,2′-anhydronucleosides, 1a and 1b. Upon storage of 9a,b at man temperature, they are quantitatively converted into 1a,b. Mild alkaline hydrolysis of 1a,b afforded their corresponding arabino nucleosides 10a,b.     

C-Nucleoside Analogues of Nicotinamide Mononucleotide (NMN)

Abstract

5-(β-D-Ribofuranosyl)nicotinamide (lie) and 6-(β-D-ribo-furanosyDpicolinamide (IId) and their corresponding o-isomers (III) were synthesized from ribonolactone. The C-nucleoside lie was further converted to its 5′-monophosphate Up which is isosteric to NMN Ip).     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Dynamics of subgenomic hepatitis C virus replicon RNA levels in Huh-7 cells after exposure to nucleoside antimetabolites

Treatment with antimetabolites results in chemically induced low nucleoside triphosphate pools and cell cycle arrest in exponentially growing cells. Since steady-state levels of hepatitis C virus (HCV) replicon RNA were shown to be dependent on exponential growth of Huh-7 cells, the effects of antimetabolites for several nucleoside biosynthesis pathways on cell growth and HCV RNA levels were investigated. A specific anti-HCV replicon effect was defined as (i). minimal interference with the exponential cell growth, (ii). minimal reduction in cellular host RNA levels, and (iii). reduction of the HCV RNA copy number per cell compared to that of the untreated control. While most antimetabolites caused a cytostatic effect on cell growth, only inhibitors of the de novo pyrimidine ribonucleoside biosynthesis mimicked observations seen in confluent replicon cells, i.e., cytostasis combined with a sharp decrease in replicon copy number per cell. These results suggest that high levels of CTP and UTP are critical parameters for maintaining the steady-state level replication of HCV replicon in Huh-7 cells.     

Raman spectroscopy of uracil DNA glycosylase-DNA complexes: insights into DNA damage recognition and catalysis

Using off-resonance Raman spectroscopy, we have examined each complex along the catalytic pathway of the DNA repair enzyme uracil DNA glycosylase (UDG). The binding of undamaged DNA to UDG results in decreased intensity of the DNA Raman bands, which can be attributed to an increased level of base stacking, with little perturbation in the vibrational modes of the DNA backbone. A specific complex between UDG and duplex DNA containing 2'-beta-fluorodeoxyuridine shows similar increases in the level of DNA base stacking, but also a substrate-directed conformational change in UDG that is not observed with undamaged DNA, consistent with an induced-fit mechanism for damage site recognition. The similar increases in the level of DNA base stacking for the nonspecific and specific complexes suggest a common enzyme-induced distortion in the DNA, potentially DNA bending. The difference spectrum of the extrahelical uracil base in the substrate-analogue complexes reveals only a small electron density reorganization in the uracil ring for the ground state complex, but large 34 cm(-)(1) downshifts in the carbonyl normal modes. Thus, UDG activates the uracil ring in the ground state mainly through H bonds to its C=O groups, without destroying its quasi-aromaticity. This result is at variance with the conclusion from a recent crystal structure, in which the UDG active site significantly distorts the flipped-out pseudouridine analogue such that a change in hybridization at C1 occurs [Parikh, S. S., et al. (2000) Proc. Natl. Acad. Sci. USA 97, 5083]. The Raman vibrational signature of the bound uracil product differs significantly from that of free uracil at neutral pH, and indicates that the uracil is anionic. This is consistent with recent NMR results, which established that the enzyme stabilizes the uracil anion leaving group by 3.4 pK(a) units compared to aqueous solution, contributing significantly to catalysis. These observations are generally not apparent from the high-resolution crystal structures of UDG and its complexes with DNA; thus, Raman spectroscopy can provide unique and valuable insights into the nature of enzyme-DNA interactions.     

Rational design and synthesis of novel nucleotide anti-Giardia agents

Design and synthesis of a novel nucleotide anti-Giardia agent that is micromolar inhibitor of Giardia trophozoite growth in culture is described.     

Synthesis and anti-viral activity of a series of d- and l-2'-deoxy-2'-fluororibonucleosides in the subgenomic HCV replicon system

Based on the discovery of (2'R)-d-2'-deoxy-2'-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of d- and l-2'-deoxy-2'-fluororibonucleosides with modifications at 5- and/or 4-positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The key step in the synthesis, the introduction of 2'-fluoro group, was achieved by either fluorination of 2,2'-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compound, namely (2'R)-d-2'-deoxy-2',5-difluorocytidine (13), demonstrated potent anti-HCV activity and toxicity to ribosomal RNA. The replacement of the 4-amino group with a thiol group resulted in the loss of activity, while the 4-methylthio substituted analogue (25) exhibited inhibition of ribosomal RNA. As N(4)-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, we combined the two functionalities of the N(4)-hydroxyl and the 2'-fluoro into one molecule, resulting (2'R)-d-2'-deoxy-2'-fluoro-N(4)-hydroxycytidine (23). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the l-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot always predict anti-HCV activity.     

Synthesis and in vitro anti-HCV activity of beta-D- and 1-2'-deoxy-2'-fluororibonucleosides

Based on the discovery of beta-D-2'-deoxy-2'-fluorocytidine as a potent anti-hepatitis C virus (HCV) agent, a series of beta-D- and L-2'-deoxy-2'-fluoroibonucleosides with modifications at 5 and/or 4 positions were synthesized and evaluated for their in vitro activity against HCV and bovine viral diarrhea virus (BVDV). The introduction of the 2'-fluoro group was achieved by either fluorination of 2,2'-anhydronucleosides with hydrogen fluoride-pyridine or potassium fluoride, or a fluorination of arabinonucleosides with DAST. Among the 27 analogues synthesized, only the 5-fluoro compounds, namely beta-D-2'-deoxy-2',5-difluorocytidine (5), had anti-HCV activity in the subgenomic HCV replicon cell line, and inhibitory activity against ribosomal RNA. As beta-D-N4-hydroxycytidine (NHC) had previously shown potent anti-HCV activity, the two functionalities of the N4-hydroxyl and the 2'-fluoro were combined into one molecule, yielding beta-D-2'-deoxy-2'-fluoro-N4-hydroxycytidine (12). However, this nucleoside showed neither anti-HCV activity nor toxicity. All the L-forms of the analogues were devoid of anti-HCV activity. None of the compounds showed anti-BVDV activity, suggesting that the BVDV system cannot reliably predict anti-HCV activity in vitro.