S--vinyl homocysteine, an analog of ethionine that is highly mutagenic for S. typhimurium TA100.

Two types of bovine pituitary gland polyprenols were resolved by silica gel chromatography; i. e., the high molecular weight dolichols of 17 to 23 isoprene units with the OH-terminal isoprene residue saturated, and a fully unsaturated decaprenol. The latter compound was found to be a mixture of molecules differing in the proportion of $\text{cis}$- and $\text{trans}$- isoprene units.     

Insulin secretion: Combined effects of phorbol ester and A23187

The effect of the ionophore, A23187, and/or the phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), on insulin secretion were compared with those of glucose. Glucose induces a biphasic pattern of insulin secretion; A23187 a comparable initial spike but no second phase; and TPA a slowly progressive increase. Combined A23187 and TPA evoke a pattern similar to that induced by glucose. Forskolin enhances both phases of glucose-induced and of TPA-A23187-induced insulin secretion. These results are interpreted in terms of a model of cell activation in which two branches of the calcium messenger system, the calmodulin branch and the C-kinase branch, control, respectively, the initial and sustained phases of insulin secretion.     

Phorbol ester inhibits 13-cis-retinoic acid-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate in cultured murine keratinocytes: a possible negative feedback via protein kinase C-activation.

The incubation of double-labelled [( 14C]-glycerol and [3H]-myoinositol) keratinocytes with 13-cis retinoic acid induced the transient and simultaneous release of [3H]-inositol trisphosphate ([3H]-InsP3) and [14C]-diacylglycerol ([14C]-DAG) indicating that a possible mode of action of this retinoid on murine keratinocytes may be at least in part the early transient release of the two putative messengers (InsP3 and DAG) from phosphatidylinositol-4,5 bisphosphate (PtdIns4, 5P2). In contrast, the preincubation of the keratinocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA) prior to incubation with 13-cis-RA suppressed the 13-cis-RA-induced release of [3H]-InsP3 and [14C]-DAG. The specificity of the TPA effect was established by the lack of effect of the biologically inactive 4 alpha-phorbol 12, 13-didecanoate. Furthermore, the incubation of the TPA-primed keratinocytes with 13-cis-RA caused a delayed and sustained accumulation of [14C]-DAG. An exploration of the source of this late release of [14C]-DAG revealed that this [14C]-DAG was released from non-inositol containing phospholipids, particularly, phosphatidylcholine. This latter DAG released in the TPA-primed cells correlated with the translocation of the cytoplasmic protein kinase C (PKC) activity to the membrane associated PKC activity. Taken together, these results suggest that alteration of PKC activity, presumably induced by DAG released from non-inositol phospholipids, may play a major role in the TPA-induced negative feedback inhibition of 13-cis RA-induced hydrolysis of keratinocyte PtdIns4, 5P2.     

Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells

Mammalian cells were after irradiation suspended in melted agarose, and casted on microscope slides. The slides were after gelling at 0°C immersed in a neutral detergent solution which lysed the cells. A weak electric field (5 V/cm) was then applied over the gel for 5 minutes. The DNA in the gel was stained with the fluorescent dye acridine orange and gives a green emission in a microscope photometer. DNA had migrated towards the anode and this migration was more pronounced in irradiated than in control cells. The differences in migration pattern were quantitatively measured. The lower detection limit was below 0.5 Gy and a plateau in the dose-effect curve was reached at about 3 Gy. In repair experiments residual DNA damage could be observed after postirradiation incubation for 60 minutes.The advantages of the method is: no radioactive labelling and only a few number of cells is required.     

Aldosterone secretion: effect of phorbol ester and A23187

The effects of the divalent ionophore, A23187, the phorbol ester, and/or 12-0-tetradecanoyl-phorbol-13-acetate on aldosterone secretion from adrenal glomerulosa cells were compared to those of angiotensin II (AII). AII causes a prompt and sustained increase in secretion. A23187 causes an initial increase followed by a gradual decline to values less than 25 percent of those seen with AII. TPA causes no initial increase but a slowly progressive rise in secretion rate to a less than maximal value. When TPA and A23187 act together, there is a prompt and sustained increase in aldosterone production rate similar to that seen after AII addition. The effect of TPA is dependent on the free Ca2+ concentration of the cell cytosol. These results are interpreted in terms of a model of cell activation in which two branches of the calcium messenger system operate to control respectively the initial and sustained phases of the secretory response. The first phase occurs as a consequence of amplitude modulation of the calmodulin branch of the system by a rise in [Ca2+]c, and the second phase as a consequence of the sensitivity modulation of the C-kinase branch by diacylglycerol.     

2-Arachidonoylglycerol, an endogenous cannabinoid receptor ligand, enhances the adhesion of HL-60 cells differentiated into macrophage-like cells and human peripheral blood monocytes

2-Arachidonoylglycerol (2-AG), an endogenous cannabionoid receptor (CB1 and CB2) ligand, enhanced the adhesion of HL-60 cells differentiated into macrophage-like cells to fibronectin and the vascular cell adhesion molecule-1. The CB2 receptor, Gi/Go, intracellular free Ca(2+) and phosphatidylinositol 3-kinase were shown to be involved in 2-AG-induced augmented cell adhesion. 2-AG also enhanced the adhesion of human monocytic leukemia U937 cells and peripheral blood monocytes. These results strongly suggest that 2-AG plays some essential role in inflammatory reactions and immune responses by inducing robust adhesion to extracellular matrix proteins and adhesion molecules in several types of inflammatory cells and immune-competent cells.     

Pertussis toxin-sensitive modulation of glutamate transport by endothelin-1 type A receptors in glioma cells

Endothelin-1 (ET-1) is a 21 amino acids peptide that exerts several biological activities through interaction with specific G-protein coupled receptors. Increased ET-1 expression is frequently associated with pathological situations involving alterations in glutamate levels. In the present study, a brief exposure to ET-1 was found to increase aspartate uptake in C6 glioma cells, which endogenously express the neuronal glutamate transporter EAAC1 (pEC₅₀ of 9.89). The stimulatory effect of ET-1 mediated by ET_A receptors corresponds to a 62% increase in the V_max with no modification of the affinity for the substrate. While protein kinase C activity is known to participate in the regulation of EAAC1, the effect of ET-1 on the glutamate uptake was found to be independent of this kinase activation. In contrast, the inactivation of G_o/i type G-protein dependent signaling with pertussis toxin was found to impair ET-1-mediated regulation of EAAC1. An examination of the cell surface expression of EAAC1 by protein biotinylation studies or by confocal analysis of immuno-fluorescence staining demonstrated that ET-1 stimulates EAAC1 translocation to the cell surface. Hence, the disruption of the cytoskeleton with cytochalasin D prevented ET-1-stimulated aspartate uptake. Together, the data presented in the current study suggest that ET-1 participates in the acute regulation of glutamate transport in glioma cells. Considering the documented role of glutamate excitotoxicity in the development of brain tumors, endothelinergic system constitutes a putative target for the pharmacological control of glutamate transmission at the vicinity of glioma cells.     

The resetting of the circadian rhythm by Prostaglandin J2 is distinctly phase-dependent

The circadian rhythm can be reset by a variety of substances. Prostaglandin J₂ (PGJ₂) is one such substance and resets the circadian rhythm in fibroblasts. In our current study, we examined the phase-dependent phase shift following PGJ₂ treatment using a real-time luciferase luminescence monitoring system. In the phase response curves, we observed 12 h differences in the times of peaks in comparison with the same analysis for forskolin. Quantification of clock gene mRNAs following PGJ₂ administration additionally revealed a rapid decrease in the Per1, Rev-erbAα and Dbp levels. Our current findings thus suggest that PGJ₂ resets the peripheral circadian clock via a mechanism that is distinct from that used by forskolin (FK).     

Dimethyl sulfoxide as an inducer of differentiation in preosteoblast MC3T3-E1 cells

Osteoblastic differentiation is an essential part of bone formation. Dimethyl sulfoxide (DMSO) is a water miscible solvent that is used extensively for receptor ligands in osteoblast studies. However, little is known about its effects on osteoblastogenic precursor cells. In this study, we have used a murine preosteoblast cell line MC3T3-E1 cells to demonstrate that DMSO effectively induces osteoblastic differentiation of MC3T3-E1 cells via the activation of Runx2 and osterix and is dependent upon the protein kinase C (PKC) pathways. We further demonstrated that prolonged activation of PKC pathways is sufficient to induce osteoblastic differentiation, possibly via the activation of PKD/PKCmu.     

Common and distinct roles for the binding partners Rabenosyn-5 and Vps45 in the regulation of endocytic trafficking in mammalian cells

In several invertebrate organisms, the Sec1p/Munc18-like protein Vps45 interacts with the divalent Rab4/Rab5 effector, Rabenosyn-5 and carries out multiple functions in the endocytic/secretory pathways. In mammalian cells, Vps45 and Rabenosyn-5 also interact, but the molecular characterization of this binding, and the functional relationship between these two proteins has not been well defined. Here we identify a novel sequence within Rabenosyn-5 required for its interaction with Vps45. We demonstrate that hVps45-depletion decreases expression of Rabenosyn-5, likely resulting from Rabenosyn-5 degradation through the proteasomal pathway. Furthermore, we demonstrate that similar to Rabenosyn-5-depletion, hVps45-depletion causes impaired recycling of β1 integrins, and a subsequent delay in human fibroblast cell migration on fibronectin-coated plates. Moreover, β1 integrin recycling could be rescued by reintroduction of siRNA-resistant wild-type Rabenosyn-5, but not a mutant deficient in Vps45 binding. However, unlike Rabenosyn-5-depletion, which induces Golgi fragmentation and decreased recruitment of sorting nexin retromer subunits to the Golgi, hVps45-depletion induces Golgi condensation and accumulation of retromer subunits in the vicinity of the Golgi. In part, these phenomena could be attributed to reduced Syntaxin16 expression and altered localization of both Syntaxin16 and Syntaxin6 upon Vps45-depletion. Overall, these findings implicate hVps45 and Rabenosyn-5 in post early endosome transport, and we propose that their interaction serves as a nexus to promote bidirectional transport along the endosome-to-recycling compartment and endosome-to-Golgi axes.     

Design,synthesis, and biological evaluation of some novel 4-aminoquinazolines as Pan-PI3K inhibitors

A series of 4-aminoquinazolines derivatives containing hydrophilic group were designed and identified as potent Pan-PI3K inhibitors in this study. The results of antiproliferative assays in vitro showed that this series of compounds had strong inhibition of tumor growth, especially compound 7b for MCF-7 cells but weak inhibition to normal cells. PI3K kinase assay showed that 7b had high activity for three PI3K isoforms with the IC₅₀ values of picomole. The western blot assay indicated that 7b could decrease the phospho-Akt (S473) in a dose-dependent manner. Further experiments showed that 7b could induce apoptosis in MCF-7 cells. Four key hydrogen bonding interactions were found in the docking of 7b with PI3K kinase. All these results suggested that 7b is a potent PI3K inhibitor and could be considered as a potential candidate for the development of anticancer agents.     

Removal or masking of phosphatidylinositol(4,5)bisphosphate from the outer mitochondrial membrane causes mitochondrial fragmentation

Mitochondria are central players in programmed cell death and autophagy. While phosphoinositides are well established regulators of membrane traffic, cellular signalling and the destiny of certain organelles, their presence and role for mitochondria remain elusive. In this study we show that removal of PtdIns(4,5)P₂ by phosphatases or masking the lipid with PH domains leads to fission of mitochondria and increased autophagy. Induction of general autophagy by amino acid starvation also coincides with the loss of mitochondrial PtdIns(4,5)P₂, suggesting an important role for this lipid in the processes that govern mitophagy. Our findings reveal that PKCα can rescue the removal or masking of PtdIns(4,5)P₂, indicating that the inositol lipid is upstream of PKC.     

Effect of Serine Phosphorylation and Ser25 Phospho-Mimicking Mutations on Nuclear Localisation and Ligand Interactions of Annexin A2

Annexin A2 (AnxA2) interacts with numerous ligands, including calcium, lipids, mRNAs and intracellular and extracellular proteins. Different post-translational modifications participate in the discrimination of the functions of AnxA2 by modulating its ligand interactions. Here, phospho-mimicking mutants (AnxA2-S25E and AnxA2-S25D) were employed to investigate the effects of Ser25 phosphorylation on the structure and function of AnxA2 by using AnxA2-S25A as a control. The overall α-helical structure of AnxA2 is not affected by the mutations, since the thermal stabilities and aggregation tendencies of the mutants differ only slightly from the wild-type (wt) protein. Unlike wt AnxA2, all mutants bind the anxA2 3′ untranslated region and β-γ-G-actin with high affinity in a Ca^2 +-independent manner. AnxA2-S25E is not targeted to the nucleus in transfected PC12 cells. In vitro phosphorylation of AnxA2 by protein kinase C increases its affinity to mRNA and inhibits its nuclear localisation, in accordance with the data obtained with the phospho-mimicking mutants. Ca^2 +-dependent binding of wt AnxA2 to phosphatidylinositol, phosphatidylinositol-3-phosphate, phosphatidylinositol-4-phosphate and phosphatidylinositol-5-phosphate, as well as weaker but still Ca^2 +-dependent binding to phosphatidylserine and phosphatidylinositol-3,5-bisphosphate, was demonstrated by a protein–lipid overlay assay, whereas binding of AnxA2 to these lipids, as well as its binding to liposomes, is inhibited by the Ser25 mutations. Thus, introduction of a modification (mutation or phosphorylation) at Ser25 appears to induce a conformational change leading to increased accessibility of the mRNA- and G-actin-binding sites in domain IV independent of Ca^2 + levels, while the Ca^2 +-dependent binding of AnxA2 to phospholipids is attenuated.     

The in vitro and in vivo apoptotic effects of Mahonia oiwakensis on human lung cancer cells

Both the root and stem bark of Mahonia species were popular folk medicines. The plant has several proven biological activities including anti-bacterial, anti-fungal, and anti-inflammatory effects. However, Mahonia has not been studied for its anticancer effects. In the present study, we made extracts from Mahonia oiwakensis (MOE), a selected species in Taiwan, and investigated their effects on various human lung cells. We found that MOE-induced apoptotic death in human A549 non-small-cell lung carcinoma (NSCLC) cells in a dose- and time-dependent manner. Treatment with the extracts also caused an increase in the sub-G1 fraction of cells, chromosome condensation, and DNA fragmentation. The mitochondrial-mediated pathway was implicated in this MOE-induced apoptosis as evidenced by the activation of the caspase cascade, cleavage of poly (ADP-ribose) polymerase (PARP), disruption of mitochondrial membrane potential, and release of cytochrome C. A higher ratio of Bax/Bcl-2 proteins and cleavage of Bid were also observed in MOE-induced cell apoptosis. In A549 tumor-xenografted nude mice, MOE also retarded in vivo proliferation (P < 0.05) and induced apoptosis in tumor cells, as shown by a decrease in Ki-67-positive staining (P < 0.05) and increased transferase-mediated dUTP nick-end labeling (TUNEL)-positive staining (P < 0.05). In conclusion, MOE inhibits the growth of human lung cancer cells in vitro and in vivo, suggesting that it may have therapeutic potential against human lung cancer.     

Transcriptional suppression of nephrin in podocytes by macrophages: roles of inflammatory cytokines and involvement of the PI3K/Akt pathway

Expression of nephrin, a crucial component of the glomerular slit diaphragm, is downregulated in patients with proteinuric glomerular diseases. Using conditionally immortalized reporter podocytes, we found that bystander macrophages as well as macrophage-derived cytokines IL-1beta and TNF-alpha markedly suppressed activity of the nephrin gene promoter in podocytes. The cytokine-initiated repression was reversible, observed on both basal and inducible expression, independent of Wilms' tumor suppressor WT1, and caused in part via activation of the phosphatidylinositol-3-kinase/Akt pathway. These results indicated a novel mechanism by which activated macrophages participate in the induction of proteinuria in glomerular diseases.     

Design, synthesis, and biological evaluation of N-acetyl-S-(p-chlorophenylcarbamoyl)cysteine and its analogs as a novel class of anticancer agents

N-Acetyl-S-(p-chlorophenylcarbamoyl)cysteine (NACC) was identified as a metabolite of sulofenur. Sulofenur was demonstrated to have broad activity against solid tumors in preclinical studies but exhibited disappointing clinical responses due to its high protein binding related adverse effects. NACC exhibited low protein binding and excellent activity against a sulofenur sensitive human colon cancer cell line. In this study, analogs of NACC were synthesized and evaluated with four human cancer cell lines. Two of the NACC analogs showed excellent activity against two human melanoma cell lines, while NACC remains the most potent of the series. All three compounds were more potent than dacarbazine, which is used extensively in treating melanoma. NACC was shown to induce apoptosis without affecting the cell cycle. Further, NACC exhibited low toxicity against monkey kidney cells. The selective anticancer activity, low toxicity, an unknown yet but unique anticancer mechanism and ready obtainability through synthesis make NACC and its analogs promising anticancer agents.