Biosynthesis of the tigloyl esters in Datura: The role of 2-methylbutyric acid

Datura innoxia plants were wick fed with (±)-2-methylbutyric acid-[1-¹⁴C] and harvested after 7 days. The root alkaloids 3α,6β-ditigloyloxytropane and 3α,6β-ditigloyloxytropan-7β-ol were isolated and degraded. In each case the radioactivity was located in the ester carbonyl group indicating that this acid is an intermediate in the biosynthesis of tiglic acid from l-isoleucine. On the other hand, (±)-2-hydroxy-2-methylbutyric acid-[1-¹⁴C], which was fed to hydroponic cultures of Datura innoxia alongside isoleucine[U-¹⁴C] positive control plants, is not an intermediate.     

Neolignans from an Aniba species

A benzene extract of the trunk wood of an Aniba species contained 3a-allyl-2-aryl-5-methoxy-3-methyl-2,3,3a,6-tetrahydro-6-oxobenzofurans which may be responsible, through sequential Cope, retro-Claisen and Claisen rearrangements respectively for the formation of the co-occurring 5-allyl-2-aryl-5-methoxy-3-methyl-2,3,5,6-tetrahydro-6-oxobenzofurans; the 6-O-allyl-2-aryl-5-methoxy-3-methyl-2,3-dihydrobenzofurans and the 7-allyl-2-aryl-6-hydroxy-5-methoxy-3-methyl-2,3-dihydrobenzofurans. The examination of the stereochemistry of these products led to the formulation of burchellin, previously isolated from Aniba burchellii Kostermans, as (2S,3S,3aR)-3a-allyl-5-methoxy-2-piperonyl-3-methyl-2,3,3a,6-tetrahydro-6-oxobenzofuran. The structure 1-allyl-4,8-dihydroxy-7-(3-methoxy-4,5-methylenedioxyphenyl)-6-methyl-3-oxobicyclo[3,2,1]octane is tentatively proposed for an additional neolignan.     

Biosynthesis of the tigloyl esters of Datura: Cis-trans isomerism

Datura innoxia plants were wick fed with angelic acid-[1-¹⁴C] and l-isoleucine-[U-¹⁴C] to act as a positive control. After 7 days the root alkaloids 3α-tigloyloxytropane, 3α,6β-ditigloyloxytropane, and 3α,6β-ditigloyloxytropan-7β-ol were isolated and it was determined that angelic acid is not a precursor for the tigloyl moiety of these alkaloids. Tiglic acid-[1-¹⁴C] which was fed via the roots to hydroponic cultures of Datura innoxia, was incorporated to a considerable degree after 8 days.     

The formation of methylated bases in DNA by dimethylnitrosamine and its relation to differences in the formation of tumours in the livers of GR and C3HF mice

Metabolism of and DNA methylation by dimethylnitrosamine (DMNA) were measured in the livers of GR male and C3Hf male and female mice which showed widely different susceptibilities to tumour formation by this hepatocarcinogen.It was previously shown that continuous DMNA administration results in vascular tumours in the livers of C3Hf female mice, whereas C3Hf males develop a high incidence of hepatomas both after continuous treatment and after a single injection of DMNA to adult animals. GR males showed a low susceptibility to the formation of liver tumours under these conditions.N-demethylation of DMNA by liver microsomes showed similar activity for both C3Hf sexes; but GR males were significantly more active.At 5 and 48 h after a single injection of [¹⁴C]DMNA, the amounts of O⁶-methylguanine (O⁶-MeGua), 7-methylguanine (7-MeGua), 1-methyladenine (1-MeAde) and 3-methyladenine (3-MeAde) were similar for C3Hf males and females, with the possible exception of 7-MeGua which seemed to be slightly higher in the female. O⁶ MeGua disappeared from C3Hf liver DNA with an apparent half-life time of about 24 h. Especially at 48 h after injection, GR liver DNA was methylated to a higher extent than was C3Hf liver DNA. This result, which antiparallels the tumour incidences, may be explained by the differences in rate of N-demethylation of DMNA. where higher 7-MeGua values were found for fasted animals under otherwise identical conditions.The general conclusior to be drawn is that neither the metabolism of DMNA nor DNA methylation by this carcinogen in the livers of male GR and C3Hf male and female mice correlates With the formation of hepatomas after DMNA administration. A possible explanation of the absence of such a correlation between DNA methylation and tumour formation might be that there exists no causal relationship between both events. However, a complicating factor is that the eventual development of a tumour may be influenced by a number of—sometimes decisive—secondary factors like hormonal²⁵ or immunological²⁶ status or the presence of cellular proliferation in target organs^27,28. Evidence from other systems suggests a relationship between inactivating, mutagenic or carcinogenic effects of alkylating agents and their ability to interact with nucleic acids, especially DNA^29,30.     

The neolignans of Licaria canella

The trunk wood of Licaria canella contains, besides dillapiol and elemicin, the neolignans canellin-A, -B and -C, for which the respective structures of 1-allyl-4,8-dihydroxy-3,5-dimethoxy-7-methyl-6-piperonylbicyclo-[3,2,1]octane; 3a-allyl-4,5-dimethoxy-3-methyl-2-piperonyl-2,3,3a,6,7,7a-hexahydro-6-oxobenzofuran and 1-allyl-4,8-dihydroxy-5-methoxy-7-methyl-6-piperonyl-3-oxobicyclo-[3,2,1] octane are proposed.     

Flavonoids from the leaf resin of Adenostoma sparsifolium

Four flavonoids were identified from the external leaf extract of Adenostoma sparsifolium: the two new flavones 3, 7-dihydroxy-5, 6-dimethoxyflavone and 3, 5, 7-trihydroxy-8-methoxyflavone and the known compounds galangin and pinocembrin.     

Cryo-EM reveals conformational flexibility in apo DNA polymerase ζ

The translesion synthesis (TLS) DNA polymerases Rev1 and Polζ function together in DNA lesion bypass during DNA replication, acting as nucleotide inserter and extender polymerases, respectively. While the structural characterization of the Saccharomyces cerevisiae Polζ in its DNA-bound state has illuminated how this enzyme synthesizes DNA, a mechanistic understanding of TLS also requires probing conformational changes associated with DNA- and Rev1 binding. Here, we used single-particle cryo-electron microscopy to determine the structure of the apo Polζ holoenzyme. We show that compared with its DNA-bound state, apo Polζ displays enhanced flexibility that correlates with concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. We also identified a lysine residue that obstructs the DNA-binding channel in apo Polζ, suggesting a gating mechanism. The Polζ subunit Rev7 is a hub protein that directly binds Rev1 and is a component of several other protein complexes such as the shieldin DNA double-strand break repair complex. We analyzed the molecular interactions of budding yeast Rev7 in the context of Polζ and those of human Rev7 in the context of shieldin using a crystal structure of Rev7 bound to a fragment of the shieldin-3 protein. Overall, our study provides new insights into Polζ mechanism of action and the manner in which Rev7 recognizes partner proteins.     

Biosynthesis of ditigloyl esters of DI- and tri-hydroxytropanes in Datura

3α-Tigloyloxytropane-[¹⁴CO] [N-¹⁴Me], ratio 1·6:1 and valtropine-[¹⁴CO] [N-¹⁴Me], ratio 1·75:1 were separately fed via cotton wicks to 4-month-old Datura innoxia plants. After 8 days the root alkaloids 3α-tigloyloxytropane, 3α,6β-ditigloyloxytropane and 3α,6β-ditigloyloxytropan-7β-ol were isolated and the distribution of radioactivity in the acid and alkamine moieties was determined by hydrolysis. The precursor ratios were not maintained in the isolated ditigloyl esters, a result which does not support our hypothesis that the ditigloyl esters are formed by the progressive hydroxylation of 3α-tigloyloxytropane.     

Benzofuranoid neolignans from Aniba simulans

Forteen neolignans, isolated from the benzene extract of Aniba simulans (Lauraceae) trunk wood, included the hitherto undescribed (2S, 3S, 5R)-5-allyl-5,7-dimethoxy-2-(3′,4′,5′-trimethoxyphenyl)-3-methyl-2,3,5,6-tetra-hydro-6-oxobenzofuran, (2R,3S,5R) -5-allyl-5-methoxy-2-(3′-methoxy-4′,5′-methylenedioxyphenyl)-3-methy1-2,3,5, 6-tetrahydro-6-oxobenzofuran, (2S,3S)-6-O-allyl -5-methoxy-2-(3′-methoxy-4′-5′-methylenedioxyphenyl)-3-methyl-2,3-dihydrobenzofuran, (2R,3S)-6-O-allyl-5-methoxy-2- (3′-methoxy-4′,5′-methylenedioxyphenyl)-3-methyl-2,3-dihydrobenzofuran and 7-allyl-6-hydroxy-5-methoxy-2-(3′-methoxy-4,5′ -methylenedioxyphenyl)-3-methylbenzofuran.     

Minor xanthones of Hypericum mysorense

2-Hydroxyxanthone, 1,7-dihydroxyxanthone, 1-hydroxy-7-methoxyxanthone, 6,7-dimethoxy-1-hydroxyxanthone and a new natural product, 2-hydroxy-3-methoxyxanthone, have been isolated and characterized from the phenolic fraction of the chloroform extract of the timber of Hypericum mysorense. The presence of simple xanthones in this genus supports the classification of Hypericum in the subfamily Hypericoideae in Guttiferae.     

Dramatic suppression of colorectal cancer cell growth by the dual mTORC1 and mTORC2 inhibitor AZD-2014

Colorectal cancer is a major contributor of cancer-related mortality. The mammalian target or rapamycin (mTOR) signaling is frequently hyper-activated in colorectal cancers, promoting cancer progression and chemo-resistance. In the current study, we investigated the anti-colorectal cancer effect of a novel mTOR complex 1 (mTORC1) and mTORC2 dual inhibitor: AZD-2014. In cultured colorectal cancer cell lines, AZD-2014 significantly inhibited cancer cell growth without inducing significant cell apoptosis. AZD-2014 blocked activation of both mTORC1 (S6K and S6 phosphorylation) and mTORC2 (Akt Ser 473 phosphorylation), and activated autophagy in colorectal cancer cells. Meanwhile, autophagy inhibition by 3-methyaldenine (3-MA) and hydroxychloroquine, as well as by siRNA knocking down of Beclin-1 or ATG-7, inhibited AZD-2014-induced cytotoxicity, while the apoptosis inhibitor had no rescue effect. In vivo, AZD-2014 oral administration significantly inhibited the growth of HT-29 cell xenograft in SCID mice, and the mice survival was dramatically improved. At the same time, in xenografted tumors administrated with AZD-2014, the activation of mTORC1 and mTORC2 were largely inhibited, and autophagic markers were significantly increased. Thus, AZD-2014 inhibits colorectal cancer cell growth both in vivo and in vitro. Our results suggest that AZD-2014 may be further investigated for colorectal cancer therapy in clinical trials.     

Non-reversibility of the isoleucine-acetate pathway in Datura

Five-month-old Datura meteloides plants were fed via the roots with 3-hydroxy-2-methylbutanoic acid-[1-¹⁴C] and isoleucine-[U-¹⁴C] as a positive control. After 5 days the plants were collected and in each case the root alkaloids 3α,6β-ditigloyloxytropane, 3α,6β-ditigloyloxytropan-7β-ol, meteloidine, hyoscine and hyoscyamine were isolated. Whereas isoleucine served as a precursor for the tiglic acid moieties 3-hydroxy-2-methylbutanoic acid did not.     

Activation of decapping involves binding of the mRNA and facilitation of the post-binding steps by the Lsm1-7–Pat1 complex

Decapping is a critical step in the conserved 5′-to-3′ mRNA decay pathway of eukaryotes. The hetero-octameric Lsm1-7–Pat1 complex is required for normal rates of decapping in this pathway. This complex also protects the mRNA 3′-ends from trimming in vivo. To elucidate the mechanism of decapping, we analyzed multiple lsm1 mutants, lsm1-6, lsm1-8, lsm1-9, and lsm1-14, all of which are defective in decapping and 3′-end protection but unaffected in Lsm1-7–Pat1 complex integrity. The RNA binding ability of the mutant complex was found to be almost completely lost in the lsm1-8 mutant but only partially impaired in the other mutants. Importantly, overproduction of the Lsm1-9p- or Lsm1-14p-containing (but not Lsm1-8p-containing) mutant complexes in wild-type cells led to a dominant inhibition of mRNA decay. Further, the mRNA 3′-end protection defect of lsm1-9 and lsm1-14 cells, but not the lsm1-8 cells, could be partly suppressed by overproduction of the corresponding mutant complexes in those cells. These results suggest the following: (1) Decapping requires both binding of the Lsm1-7–Pat1 complex to the mRNA and facilitation of the post-binding events, while binding per se is sufficient for 3′-end protection. (2) A major block exists at the post-binding steps in the lsm1-9 and lsm1-14 mutants and at the binding step in the lsm1-8 mutant. Consistent with these ideas, the lsm1-9, 14 allele generated by combining the mutations of lsm1-9 and lsm1-14 alleles had almost fully lost the RNA binding activity of the complex and behaved like the lsm1-8 mutant.     

Hypochaerin: a new sesquiterpene lactone from Hypochaeris setosus

Hypochaeris setosus contains desacetoxymatricarin, achillin, 1-hydroxy-6β,7α,11β-H-eudesm-4-en-6,12 olide, jacquinelin and hypochaerin, a new guaianolide, established as 3-oxo-4β,5α,6β,7α,11β-H-guai-1(2)-en-6,12 olide.     

Guianin: A neolignan from Aniba guianensis

The wood of Aniba guianensis Aubl. (Lauraceae) contains benzyl benzoate, benzyl salicylate, sitosterol, O-methyleugenol, O-methylisoeugenol and the neolignan guianin for which the structure of 1-allyl-8-hydroxy-3-methoxy-7-methyl-4-oxo-6-piperonylbicyclo[3,2,1]oct-2-ene (VI) is proposed.     

The isolation and characterization of 3-(2-carboxyethyl)cytosine following in vitro reaction of β-propiolactone with calf thymus DNA

The new adduct 3-(2-carboxyethyl)cytosine (3-CEC) was isolated following in vitro reaction of the carcinogen β-propiolactone (BPL) with calf thymus DNA. The structure of 3-CEC was confirmed by synthesis from BPL and dCyd. Reaction of BPL with cCyd (pH 7.0–7.5, 37°C) gave 3-(2-carboxyethyl)deoxycytidine (3-CEdCyd) (9% yield) and 3,N⁴-bis(2-carboxyethyl)deoxycytidine (3,N⁴-BCEdCyd) (0.6% yield). 3-CEdCyd and 3,N⁴-BCEdCyd were hydrolyzed (1.5 N HC1, 100°C, 2 h) to 3-CEC and 3,N⁴-bis(2-carboxyethyl)cytosine (3,N⁴-BCEC), respectively. The structure of 3-CEC was assigned on the basis of UV and NMR spectra and the electron impact (EI) mass spectra of 3-CEC and a tri-trimethylsilyl (TMS) derivative of 3 CEC as well as deuterated (d₂₇) tri-TMS derivative of 3-CEC. The structure of 3,N⁴-BCEC was assigned on the basis of UV spectra and the EI mass spectra of a tri-TMS derivative. EI and isobutane chemical ionization mass spectra of 3-methylcytosine (3-MeCyt) and a di-TMS derivative of 3-MeCyt were obtained and were helpful in deducing the structures of 3-CEC and 3,N⁴-BCEC. This is the first report of the alkylation by BPL of an exocyclic atom on a base in DNA. Compound 3,N⁴-BCEC was not detected in BPL-reacted calf thymus DNA. The relative amounts of 1-(2-carboxyethyl)adenine (1-CEA), 7-(2-carboxyethyl)guanine (7-CEG), 3-(2-carboxyethyl)thymine (3-CET) and 3-CEC isolated from BPL-reacted DNA following perchloric acid hydrolysis were 0.23, 1.00, 0.39 and 0.41 respectively, when the alkylation reaction was conducted in phosphate buffer at 0–5°C and pH 7.5 and 0.10, 1.00, 0.29 and 0.28 respectively when the reaction was conducted in H₂O at 37°C and pH 7.0–7.5.     

Increased urinary excretion of pyrimidine and nicotinamide derivatives in rats treated with methyl methanesulphonate

Rats treated with methyl methanesulphonate (MMS) excreted significantly higher quantities of deoxycytidine, thymidine, uracil, 1-methylnicotinamide (1-meNmd) and 1-methyl-6-pyridone-3-carboxylamide (6-pyr-1-meNmd) in their urine 0–24 h after MMS injection (100 $\text{mg}\text{kg}$). Excretion of thymidine, which was not detectable in untreated rats, was dose-dependent. No increase in urinary 7-methylguanine was found, and creatinine excretion was decreased by MMS treatment. Experiments with methyl-¹⁴C-labelled MMS showed transfer of ¹⁴C-label to 7-methylguanine and 1-meNmd. X-Irradiation (500 rad) caused increased excretion of pyrimidines, like MMS, but did not increase excretion of the nicotinamide derivatives.     

Xanthones from Tovomita pyrifolium

The wood of Tovomita pyrifolium (Guttiferae) contains the novel tovopyrifolins A [1,6-dihydroxy-7-methoxy-5-prenyl-6′,6′-dimethylpyrano (2′,3′:3,2)xanthone], B (1,5-dihydroxy-3,4-dimethoxyxanthone) and C (1,3,5-trihydroxy-2-methoxyxanthone) and also the known tovophyllins A and B [structure revised to 1,6-dihydroxy-5-prenyl-6′, 6′-dimethylpyrano(2′,3′:3,2)-6″,6″-dimethylpyrano(2″,3″:7,8)xanthone].     

Increased Cytokine and Nitric Oxide Levels in Serum of Dogs Experimentally Infected with Rangelia vitalii

This study aimed to measure the levels of interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), interleukin 1 (IL-1), interleukin 6 (IL-6), and nitrite/nitrate (NO_x) in serum of dogs experimentally infected with Rangelia vitalii. Twelve female mongrel dogs were divided into 2 groups; group A (uninfected controls) composed by healthy dogs (n=5) and group B consisting of dogs inoculated with R. vitalii (n=7). Animals were monitored by blood smear examinations, which showed intraerythrocytic forms of the parasite on day 5 post-infection (PI). Blood samples were collected through the jugular vein on days 0, 10, and 20 PI to determine the serum levels of IFN-γ, TNF-α, IL-1, IL-6, and NO_x. Cytokines were assessed by ELISA quantitative sandwich technique, and NO_x was measured by the modified Griess method. Cytokine levels (IFN-γ, TNF-α, IL-1, and IL-6) were increased (P<0.01) in serum of infected animals. Serum levels of NO_x were also increased on days 10 PI (P<0.01) and 20 PI (P<0.05) in infected animals. Therefore, the infection with R. vitalii causes an increase in proinflammatory cytokines and nitric oxide content. These alterations may be associated with host immune protection against the parasite.     

HMGA1-pseudogene expression is induced in human pituitary tumors

Numerous studies have established that High Mobility Group A (HMGA) proteins play a pivotal role on the onset of human pituitary tumors. They are overexpressed in pituitary tumors, and, consistently, transgenic mice overexpressing either the Hmga1 or the Hmga2 gene develop pituitary tumors. In contrast with HMGA2, HMGA1 overexpression is not related to any rearrangement or amplification of the HMGA1 locus in these tumors. We have recently identified 2 HMGA1 pseudogenes, HMGA1P6 and HMGA1P7, acting as competitive endogenous RNA decoys for HMGA1 and other cancer related genes. Here, we show that HMGA1 pseudogene expression significantly correlates with HMGA1 mRNA levels in growth hormone and nonfunctioning pituitary adenomas likely inhibiting the repression of HMGA1 through microRNAs action. According to our functional studies, these HMGA1 pseudogenes enhance the proliferation and migration of the mouse pituitary tumor cell line, at least in part, through their upregulation. Our results point out that the overexpression of HMGA1P6 and HMGA1P7 could contribute to increase HMGA1 levels in human pituitary tumors, and then to pituitary tumorigenesis.