Phosphorylation of Mcl‐1 by CDK1–cyclin B1 initiates its Cdc20‐dependent destruction during mitotic arrest

The balance between cell cycle progression and apoptosis is important for both surveillance against genomic defects and responses to drugs that arrest the cell cycle. In this report, we show that the level of the human anti‐apoptotic protein Mcl‐1 is regulated during the cell cycle and peaks at mitosis. Mcl‐1 is phosphorylated at two sites in mitosis, Ser64 and Thr92. Phosphorylation of Thr92 by cyclin‐dependent kinase 1 (CDK1)–cyclin B1 initiates degradation of Mcl‐1 in cells arrested in mitosis by microtubule poisons. Mcl‐1 destruction during mitotic arrest requires proteasome activity and is dependent on Cdc20/Fizzy, which mediates recognition of mitotic substrates by the anaphase‐promoting complex/cyclosome (APC/C) E3 ubiquitin ligase. Stabilisation of Mcl‐1 during mitotic arrest by mutation of either Thr92 or a D‐box destruction motif inhibits the induction of apoptosis by microtubule poisons. Thus, phosphorylation of Mcl‐1 by CDK1–cyclin B1 and its APC/C^Cdc20‐mediated destruction initiates apoptosis if a cell fails to resolve mitosis. Regulation of apoptosis, therefore, is linked intrinsically to progression through mitosis and is governed by a temporal mechanism that distinguishes between normal mitosis and prolonged mitotic arrest.     

Cell‐cycle‐dependent PC‐PLC regulation by APC/CCdc20‐mediated ubiquitin‐proteasome pathway

Phosphatidylcholine‐specific phospholipase C (PC‐PLC) is involved in the cell signal transduction, cell proliferation, and apoptosis. The mechanism of its action, however, has not been fully understood, particularly, the role of PC‐PLC in the cell cycle. In the present study, we found that cell division cycle 20 homolog (Cdc20) and PC‐PLC were co‐immunoprecipitated reciprocally by either antibody in rat hepatoma cells CBRH‐7919 as well as in rat liver tissue. Using confocal microscopy, we found that PC‐PLC and Cdc20 were co‐localized in the perinuclear endoplasmic reticulum region (the “juxtanuclear quality control” compartment, JUNQ). The expression level and activities of PC‐PLC changed in a cell‐cycle‐dependent manner and were inversely correlated with the expression of Cdc20. Intriguingly, Cdc20 overexpression altered the subcellular localization and distribution of PC‐PLC, and caused PC‐PLC degradation by the ubiquitin proteasome pathway (UPP). Taken together, our data indicate that PC‐PLC regulation in cell cycles is controlled by APC/C^Cdc20‐mediated UPP. J. Cell. Biochem. 107: 686–696, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of 20‐hydroxyecdysone and juvenile hormone on octopamine metabolism in females of Drosophila

The effect of exogenous 20‐hydroxyecdysone (20E) and juvenile hormone (JH) on the activities of the tyrosine decarboxylase (TDC), the first enzyme in octopamine (OA) synthesis, has been studied in young females of wild type D. virilis and D. melanogaster under normal and heat stress (38°C) conditions. Flies fed 20E expressed increased TDC activity in both species. JH application decreased TDC activity in both species. A rise in JH and 20E levels did not prevent a TDC response to heat stress, but changed the response intensity. A long‐term increase in JH titre had no effect on the activity of main OA catabolyzing enzyme, arylalkylamine N‐acetyltransferase, in females of both species. A possible mechanism of regulation of OA levels by 20E and JH in Drosophila females is discussed. © 2009 Wiley Periodicals, Inc.     

1‐Aminobenzocyclobutene‐1‐phosphonic Acid and Related Compounds as Inhibitors of Phenylalanine Ammonia‐Lyase

Five new geminal aminocycloalkanephosphonic acids ( 4 – 8 ) containing both an aromatic ring and a cycloalkane ring were synthesized and evaluated as potential inhibitors of buckwheat phenylalanine ammonia‐lyase (PAL). Within the set of compounds which are related to 2‐aminoindane‐2‐phosphonic acid (AIP, 3 ), a known powerful inhibitor of PAL, racemic 1‐aminobenzocyclobutene‐1‐phosphonic acid ( 4 ), was six times weaker than AIP as an in vitro inhibitor of buckwheat PAL, but six times stronger than AIP as an in vivo inhibitor of phenylalanine‐derived anthocyanin synthesis in buckwheat.     

The deubiquitinase activity of A20 is dispensable for NF‐κB signaling

A20 has been suggested to limit NF‐κB activation by removing regulatory ubiquitin chains from ubiquitinated substrates. A20 is a ubiquitin‐editing enzyme that removes K63‐linked ubiquitin chains from adaptor proteins, such as RIP1, and then conjugates them to K48‐linked polyubiquitin chains to trigger proteasomal degradation. To determine the role of the deubiquitinase function of A20 in downregulating NF‐κB signaling, we have generated a knock‐in mouse that lacks the deubiquitinase function of A20 (A20‐OTU mice). These mice are normal and have no signs of inflammation, have normal proportions of B, T, dendritic, and myeloid cells, respond normally to LPS and TNF, and undergo normal NF‐κB activation. Our results thus indicate that the deubiquitinase activity of A20 is dispensable for normal NF‐κB signaling.     

Spectrofluorimetric determination of aluminium using 2‐hydroxy‐1‐naphthylidene‐(8‐aminoquinoline)

A sensitive and selective spectrofluorimetric method has been developed for the rapid determination of aluminium. This method is based on the complex formation between aluminium and 2‐hydroxy‐1‐naphthylidene‐(8‐aminoquinoline) (HNAQ). The optimum conditions for the complex formation were a metal‐to‐ligand (M : L) stoichiometric ratio of 1:1, a pH of 5.5 and a 0.20 m acetate buffer. The fluorescence of the complex was monitored at an emission wavelength of 502 nm with excitation at 438 nm. Under these conditions, linear calibration curves were obtained in the ranges 0.05–1 and 1–5 ppm. The detection limit was 3.4 ppb for the former and 13.5 ppb for the latter. The maximum relative standard deviation of the method for an aluminium standard of 200 ppb was 1.5% (n = 5). This method was successfully applied for the determination of aluminium in drinking water, pharmaceutical antacid tablets and suspension samples. Copyright © 2010 John Wiley & Sons, Ltd.     

5α‐Androst‐16‐en‐3α‐ol β‐D‐Glucuronide,Precursor of 5α‐Androst‐16‐en‐3α‐ol in Human Sweat

5α‐Androst‐16‐en‐3α‐ol (α‐androstenol) is an important contributor to human axilla sweat odor. It is assumed that α‐andostenol is excreted from the apocrine glands via a H₂O‐soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H₂O‐soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α‐androstenol, β‐androstenol sulfates, 5α‐androsta‐5,16‐dien‐3β‐ol (β‐androstadienol) sulfate, α‐androstenol β‐glucuronide, α‐androstenol α‐glucuronide, β‐androstadienol β‐glucuronide, and α‐androstenol β‐glucuronide furanose. The occurrence of α‐androstenol β‐glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative‐ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α‐androstenol was observed after incubation of the sterile human sweat or α‐androstenol β‐glucuronide with a commercial glucuronidase enzyme, the urine‐isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β‐glucuronidase activities. We demonstrated that if α‐ and β‐androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H₂O‐soluble precursor of α‐androstenol in apocrine secretion should be a β‐glucuronide.     

Synthesis and Biological Evaluation of Novel 1‐(4‐(Hydroxy(1‐oxo‐1,3‐dihydro‐2H‐inden‐2‐ylidene)methyl)phenyl)‐3‐phenylurea Derivatives

A series of novel phenylurea containing 2‐benzoylindan‐1‐one derivatives 3a  –  3j were synthesized from the reaction of phenylurea‐substituted acetophenones 1a  –  1j with phthalaldehyde 2 under mild reaction conditions in good yields. All synthesized compounds were characterized by spectroscopic methods. The obtained compounds ( 3a  –  3j ) were evaluated for anticancer activity against HeLa and C6 cell lines. Antiproliferative activity was determined by the BrdU proliferation ELISA assay, 3f and 3g were found to be most active compounds. The compounds were also screened for antimicrobial activity and all compounds showed remarkable activity against used microorganisms.     

Synthesis of 3‐Methylidene‐1‐tosyl‐2,3‐dihydroquinolin‐4(1H)‐ones as Potent Cytotoxic Agents

An efficient synthetic strategy to 3‐methylidene‐2,3‐dihydroquinolin‐4(1H)‐ones variously substituted in position 2 has been developed. The title compounds were synthesized in the reaction sequence involving reaction of diethyl methylphosphonate with methyl 2‐(tosylamino)benzoate, condensation of thus formed diethyl 2‐oxo‐2‐(2‐N‐tosylphenyl)ethylphosphonate with various aldehydes followed by successful application of the obtained 3‐(diethoxyphosphoryl)‐1,2‐dihydroquinolin‐4‐ols as Horner–Wadsworth–Emmons reagents for the olefination of formaldehyde. Also, enantioselective approach to the target compounds has been evaluated using 3‐dimenthoxyphosphoryl group as a chiral auxiliary. Single X‐ray crystal analysis of (2S)‐3‐(dimenthoxyphosphoryl)‐2‐phenyl‐1‐tosyldihydroquinolin‐4‐ol revealed the presence of strong resonance‐assisted hydrogen bond (RAHB). The obtained 3‐methylidene‐2,3‐dihydroquinolin‐4(1H)‐ones were then tested for their cytotoxic activity against two leukemia cell lines NALM‐6 and HL‐60 and a breast cancer MCF‐7 cell line. All compounds showed very high cytotoxic activity with the IC₅₀ values mostly below 1 μm in all three cancer cell lines. The selected analogs were also tested on human umbilical vein endothelial cells (HUVEC) and on human mammary gland/breast cells (MCF‐10A) to evaluate their influence on normal cells. Since one of the most serious problems in cancer chemotherapy is the development of drug resistance, the mRNA levels and activity of ABCB1 transporter considered to be the most important factor engaged in drug resistance, were evaluated in MCF‐7 cells treated with two selected analogs. Both compounds were strong ABCB1 transporter inhibitors that could prevent efflux of anticancer drugs from cancer cells.     

Developing xylem‐preferential expression of PdGA20ox1, a gibberellin 20‐oxidase 1 from Pinus densiflora,improves woody biomass production in a hybrid poplar

Woody biomass has gained popularity as an environmentally friendly, renewable and sustainable resource for liquid fuel production. Here, we demonstrate biotechnological improvement of the quantity and quality of woody biomass by employing developing xylem (DX)‐preferential production of gibberellin (GA), a phytohormone that positively regulates stem growth. First, for the proof of concept experiment, we produced transgenic Arabidopsis plants expressing GA20‐oxidase, a key enzyme in the production of bioactive GAs, from Pinus densiflora (PdGA20ox1) under the control of either a constitutive 35S promoter, designated 35S::PdGA20ox1, or a DX‐specific promoter (originated from poplar), designated DX15::PdGA20ox1. As we hypothesized, both transgenic Arabidopsis plants (35S::PdGA20ox1 and DX15::PdGA20ox1) exhibited an accelerated stem growth that resulted in a large increase of biomass, up to 300% compared to wild‐type control plants, together with increased secondary wall thickening and elongation of fibre cells. Next, we applied our concept to the production of transgenic poplar trees. Both transgenic poplar trees (35S::PdGA20ox1 and DX15::PdGA20ox1) showed dramatic increases in biomass, up to 300%, with accelerated stem growth and xylem differentiation. Cell wall monosaccharide composition analysis revealed that in both Arabidopsis and poplar, glucose and xylose contents were significantly increased. However, undesirable phenotypes of 35S::PdGA20ox1 poplar, including poor root growth and leaf development, were found. Interestingly, DX15::PdGA20ox1 poplar resulted in a reduction of undesirable phenotypes. Our results indicate that the controlled production of GAs through a tissue‐specific promoter can be utilized as an efficient biotechnological tool for producing enhanced plant biomass, minimizing unwanted effects.     

Identification of LmUAP1 as a 20‐hydroxyecdysone response gene in the chitin biosynthesis pathway from the migratory locust,Locusta migratoria

In Locusta migratoria, we found that two chitin biosynthesis genes, UDP N‐acetylglucosamine pyrophosphorylase gene LmUAP1 and chitin synthase gene LmCHS1, are expressed mainly in the integument and are responsible for cuticle formation. However, whether these genes are regulated by 20‐hydroxyecdysone (20E) is still largely unclear. Here, we showed the developmental expression pattern of LmUAP1, LmCHS1 and the corresponding 20E titer during the last instar nymph stage of locust. RNA interference (RNAi) directed toward a common region of the two isoforms of LmEcR (LmEcRcom) reduced the expression level of LmUAP1, while there was no difference in the expression of LmCHS1. Meantime, injection of 20E in vivo induced the expression of LmUAP1 but not LmCHS1. Further, we found injection‐based RNAi of LmEcRcom resulted in 100% mortality. The locusts failed to molt with no apolysis, and maintained in the nymph stage until death. In conclusion, our preliminary results indicated that LmUAP1 in the chitin biosynthesis pathway is a 20E late‐response gene and LmEcR plays an essential role in locust growth and development, which could be a good potential target for RNAi‐based pest control.     

The ubiquitin‐editing enzyme A20 requires RNF11 to downregulate NF‐κB signalling

The RING domain protein RNF11 is overexpressed in breast cancers and promotes tumour growth factor‐beta (TGF‐β) signalling. RNF11 has been proposed to regulate TGF‐β signalling by interacting with HECT‐ and SCF‐type E3 ligases; however, the role of RNF11 in other signalling pathways is poorly understood. Here, we demonstrate a novel function of RNF11 as a negative regulator of NF‐κB and jun N‐terminal kinase (JNK) signalling pathways. Knockdown of RNF11 with siRNA resulted in persistent tumour necrosis factor (TNF)‐ and lipopolysaccharide (LPS)‐mediated NF‐κB and JNK signalling. RNF11 interacted with the NF‐κB inhibitor A20 and its regulatory protein TAX1BP1 in a stimulus‐dependent manner. RNF11 negatively regulated RIP1 and TRAF6 ubiquitination upon stimulation with TNF and LPS, respectively. Furthermore, RNF11 was required for A20 to interact with and inactivate RIP1 to inhibit TNF‐mediated NF‐κB activation. Our studies reveal that RNF11, together with TAX1BP1 and Itch, is an essential component of an A20 ubiquitin‐editing protein complex that ensures transient activation of inflammatory signalling pathways.     

Field evaluation on the synergistic attractiveness of 2‐(1‐undecyloxy)‐1‐ethanol and ipsenol to Monochamus saltuarius

To develop an optimal attractant for Monochamus saltuarius (Gebler) (Coleoptera: Cerambycidae), the synergistic effects of a few potential attractants (ethanol and α‐pinene as host‐plant volatiles, and ipsenol and ipsdienol as bark beetle pheromones) were tested in a pine forest combined with 2‐(1‐undecyloxy)‐1‐ethanol (monochamol), the aggregation pheromone of Monochamus species, for two consecutive years, 2014 and 2015. Total number of catches was 65 and 33 in 2014 and 2015, respectively. Ethanol or ethanol + monochamol (a base blend) were not attractive to M. saltuarius with no difference from the control. Addition of α‐pinene and ipsdienol to the base blend did not significantly increase catches. However, ipsenol was significantly synergistic to the base blend in attracting M. saltuarius in 2014, and the blend (ipsenol + base blend) attracted meaningfully higher numbers of M. saltuarius in 2015. Our study illustrates the potential for monochamol and ipsenol baits for monitoring and trapping of M. saltuarius in the field.     

Asymmetric Allylation of α‐Ketoester‐Derived N‐Benzoylhydrazones Promoted by Chiral Sulfoxides/N‐Oxides Lewis Bases: Highly Enantioselective Synthesis of Quaternary α‐Substituted α‐Allyl‐α‐Amino Acids

Chiral sulfoxides/N‐oxides (R)‐ 1 and (R,R)‐ 2 are effective chiral promoters in the enantioselective allylation of α‐keto ester N‐benzoylhydrazone derivatives 3a , 3b , 3c , 3d , 3e , 3f , 3g to generate the corresponding N‐benzoylhydrazine derivatives 4a , 4b , 4c , 4d , 4e , 4f , 4g , with enantiomeric excesses as high as 98%. Representative hydrazine derivatives 4a , 4b were subsequently treated with SmI₂, and the resulting amino esters 5a , 5b with LiOH to obtain quaternary α‐substituted α‐allyl α‐amino acids 6a , 6b , whose absolute configuration was assigned as (S), with fundament on chemical correlation and electronic circular dichroism (ECD) data. Chirality 25:529–540, 2013. © 2013 Wiley Periodicals, Inc.