Human Pancreatic Tumor Organoids Reveal Loss of Stem Cell Niche Factor Dependence during Disease Progression

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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.     

Purification and properties of a specific primase-stimulating factor of bovine thymus

The DNA replicase activity of the complex between bovine thymus DNA polymerase alpha and RNA primase was markedly decreased after the purification by ssDNA-cellulose column chromatography. In an attempt to restore the activity by supplementing some fractions eliminated from the purified enzyme, we found that a fraction eluted from the column by increasing salt concentration and 30% ammonium sulfate precipitates of the phosphocellulose-step enzyme possessed a high ability to restore the replicase activity. Thus, the factors were purified to near homogeneity from the two sources and the properties were examined. Both factors were heat-labile and trypsin-sensitive, possessed a native molecular mass of approximately 150-200 kDa as judged by Sephacryl S-200 column chromatography, and were composed of two polypeptides of 146 kDa and 47 kDa on SDS/polyacrylamide gel electrophoresis, indicating that they were an identical protein. The factor, which did not show any DNA polymerase or primase activities by itself, stimulated approximately 20-fold the replicase activity of purified DNA-polymerase-alpha-primase at a very low concentration (10 ng/50 microliter). The factor did not affect the deoxyribonucleotide polymerizing activity of the enzyme complex at all, but specifically stimulated the primase activity only. Thus, we designated the factor as primase-stimulating factor. Although varying the template concentration did not significantly affect the mode of stimulation, increasing the concentration of substrate for primer synthesis (ATP) markedly decreased the extent of stimulation. Thus, the stimulating factor seems to decrease the substrate concentration required for the primase reaction as well as increasing threefold the maximum activity attained by varying the substrate concentration. So far, no ATPase activity has been detected in the factor.     

Block of acid secretion by amytal and its partial reversal by menadione with ascorbate in the gastric mucosa of the guinea-pig

The effect of amytal on energy metabolism and acid secretion in an isolated gastric mucosa of the guinea-pig were studied. Determination of adenine nucleotides, creatine phosphate, pyruvate and lactate in the gastric mucosa showed that amytal depressed the levels of ATP, creatine phosphate and energy charge with elevation of the AMP and pyruvate levels. This treatment inhibited concomitantly acid secretion and active chloride transport detected by short circuit current. The addition of menadione with ascorbate to the medium in the presence of amytal partially restored ATP and energy charge levels and also induced a partial recovery of acid secretion and active chloride transport. These results suggest that ATP is a direct energy donor for acid secretion in the gastric mucosa of the guinea-pig.     

The African wave-type electric fish,Gymnarchus niloticus,lacks corollary discharge mechanisms for electrosensory gating

Gymnarchus niloticus, a wave-type African electric fish, performs its jamming avoidance response by relying solely upon afferent signals and does not use corollary discharges from the pacemaker nucleus in the medulla which generates the rhythmicity of electric organ discharges. This is in sharp contrast to the mode of sensory processing found in closely related African pulse-type electric fishes where afferent signals are gated by corollary discharges from the pacemaker for the distinction of exafferent and reafferent stimuli. Does Gymnarchus still possess a corollary discharge mechanism for other behavioral tasks but does not use it for the jamming avoidance response? In this study, I recorded from and labeled medullary neuronal structures that either generate or convey the pacemaker signal for electric organ discharges to examine whether this information is also sent directly to any sensory areas. The pacemaker nucleus was identified as the site of generation of the pacemaking signal. The pacemaker neurons project exclusively to the lateral relay nucleus which, in turn projects exclusively to the medial relay nucleus. Neurons in the medial relay nucleus send unbranched axons to the spinal electromotoneurons. These neurons are entirely devoted to drive the electric organ discharges, and no axon collaterals from these neurons were found to project to any sensory areas. This indicates that Gymnarchus does not possess the neuronal hardware for a corollary discharge mechanism.     

Epithelial–mesenchymal transition of A549 cells is enhanced by co-cultured with THP-1 macrophages under hypoxic conditions

Epithelial–mesenchymal transition (EMT) is associated with pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF). In this study, we investigated EMT of human pulmonary epithelial-derived cells (A549). A549 cells was either cultured by itself or co-cultured with THP-1 macrophages under normoxic (21% O₂) and hypoxic (2% O₂) conditions. We evaluated the presence of EMT by determining the expression of EMT markers, E-cadherin, vimentin, and fibronectin. To determine the role of TGF-β1 and IL-1β in EMT of the A549 cells, we analyzed the effects of blocking their activity with TGF-β1 inhibitor or IL-1β neutralizing antibody respectively. The A549 cells presented EMT when they were co-cultured with THP-1 macrophages. The EMT of the A549 cells co-cultured with THP-1 macrophages was exacerbated under hypoxia. In addition, the EMT were prevented by the addition of TGF-β1 type I receptor kinase inhibitor. The hypoxic condition increased the mRNA levels of TGF-β1 in A549 cells and THP-1 macrophages and that of IL-1β in THP-1 macrophages when each cells were co-cultured. Anti-IL-1β neutralizing antibody attenuated TGF-β1 secretion in co-culture media under hypoxic conditions. Thus, the IL-1β from THP-1 macrophages up-regulated the TGF-β1 from A549 cells and THP-1 macrophages, and then the TGF-β1 from both cells induced and promoted the EMT of A549 cells when they were co-cultured under hypoxia. Together, these results demonstrate that the interaction between type II pneumocytes and macrophages under hypoxia is necessary for the development of pulmonary fibrosis.     

Effect of Active Oxygen Radicals on Protein and Carbohydrate Moieties of Recombinant Human Erythropoietin

Our previous study showed that active oxygen radicals generated from a Fenton system and a xanthine plus xanthine oxidase system caused serious loss of in vivo bioactivity of recombinant human erythropoietin (EPO), a highly glycosylated protein. In the present study, we characterized the oxidative modifications to the protein and carbohydrate moiety of EPO, which lead to a reduction of its bioactivity. In vitro bioactivity was reduced when EPO was treated with oxygen radi cals generated from a Fenton system in the presence of 0.016 mM H₂0₂, and the reduction was directly proportional to the loss of in vivo bioactivity. SDS-PAGE analysis showed that dimer formation and degradation was observed under more severe conditions (Fenton reaction with 0.16 mM H₂0₂). The tryptophan destruction was detected at 0.016 mM H₂O₂ and well correlated with the loss of in vitro bioactivity, whereas loss of other amino acids were occurred under more severe conditions. Treatment with the Fenton system did not result in any specific damage on the carbohydrate moiety of EPO, except a reduction of sialic acid content under severe condition. These results suggest that active oxygen radicals mainly react with the protein moiety rather than the carbohydrate moiety of EPO. Destruction of tryptophan residues is the most sensitive marker of oxidative damage to EPO, suggesting the importance of tryptophan in the active EPO structure. Deglycosylation of EPO caused an increase of susceptibility to oxygen radicals compared to intact EPO. The role of oligosaccharides in EPO may be to protect the protein structure from active oxygen radicals.