Discovery of a novel Bacillus thuringiensis Cry8D protein and the unique toxicity of the Cry8D-class proteins against scarab beetles

A novel cry gene, cry8Db, highly toxic to scarab beetles such as the Japanese beetle, Popillia japonica Newman, was cloned from an isolate of Bacillus thuringiensis(Bt), BBT2-5. The cry8Db gene has 3525 bp nucleotides and codes for a protein of 1174 amino acid residues. The protein, Cry8Db, has typical Bt characteristics such as the 8-block, conserved sequences and the three-domain 3 D toxin structure as defined with Cry3Aa. When the amino acid sequence of Cry8Db was compared with that of Cry8Da whose gene was cloned and characterized in our laboratory earlier, substantial sequence diversities were found in their Domain III. The cry8Db gene was expressed in an acrystalliferous B. thuringiensis strain, BT51. BT51 expressing cry8Db formed a spherical crystal like the natural crystal of BBT2-5. The Cry8Db protein was assayed along with the other scarab active Cry8Da and Cry8Ca against the Japanese beetle. While Cry8Da and Cry8Db had toxicity against both adults and larvae of the Japanese beetle, Cry8Ca was toxic to only larvae. Cry8Ca showed no toxicity against the adult beetle up to 30 μg per 1 cm² of leaf discs on which the protein was applied. The activation process of Cry8Db by adult and larval gut juice was compared in vitro with the processes of Cry8Da and Cry8Ca. All three proteins, Cry8Db, Cry8Da and Cry8Ca, produced a toxic core of approximately 70 kDa equally indicating that the activation process does not inactivate the adult activity of Cry8Ca. We concluded that the adult activity of Cry8D proteins is encoded in Domain II. Further tests against other beetle species showed a significant difference between Cry8D’s and Cry8Ca but no difference between Cry8Da and Cry8Db. Comparison of 3D structural models of Cry8Ca, Cry8Da and Cry8Db, which were constructed by using Cry3Bb as the structural template, indicated significant structural differences, especially between Cry8Ca and Cry8D proteins, in three major surface-exposed loops of Domain II that may be involved in determining the adult beetle activity.     

New 8-hydroxyflavonoids from Solanum section Androceras

Five new 8-hydroxyflavonoids have been identified from leaves of Solanum section Androceras: 8-methoxymyricetin 3,7,4′-trimethyl ether; 8-hydroxymyricetin 3,7,4′-trimethyl ether; 8-hydroxymyricetin 8-O-glucosylxyloside 3,7,4′-trimethyl ether; 8-hydroxychrysoeriol 7-methyl ether; 8-hydroxychrysoeriol 7-O-glucoside.     

Isolation of a Bacillus thuringiensis strain (serotype 8a:8b) highly and selectively toxic against mosquito larvae

An isolate of Bacillus thuringiensis designated as PG-14 obtained from the Philippines was highly toxic to the mosquitoes Aedes aegypti and Culex molestus but nontoxic to the silkworm, Bombyx mori, and adults of a daphnid. The degree of toxicity to mosquito larvae was the same as that of the reference strain of Bacillus thuringiensis subsp. israelensis (serotype 14). Parasporal inclusion produced by the isolate PG-14 was spherical or irregular in shape and morphologically similar to that produced by the reference strain of subsp. israelensis. The H antigenic structure of the isolate was identical to that of the reference strain of B. thuringiensis subsp. morrisoni (serotype 8a:8b). Differences were shown in the O antigenic structures and in the production of lecithinase. Thermostable exotoxin was not produced by the isolate PG-14. The results indicate the isolation of a B. thuringiensis strain which shows the same toxicity as that of subsp. israelensis.     

CUX2 Protein Functions as an Accessory Factor in the Repair of Oxidative DNA Damage

CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair.     

Molecular Basis for the Antiparasitic Activity of a Mercaptoacetamide Derivative That Inhibits Histone Deacetylase 8 (HDAC8) from the Human Pathogen Schistosoma mansoni

Schistosomiasis, caused by the parasitic flatworm Schistosoma mansoni and related species, is a tropical disease that affects over 200 million people worldwide. A new approach for targeting eukaryotic parasites is to tackle their dynamic epigenetic machinery that is necessary for the extensive phenotypic changes during the life cycle of the parasite. Recently, we identified S. mansoni histone deacetylase 8 (smHDAC8) as a potential target for antiparasitic therapy. Here, we present results on the investigations of a focused set of HDAC (histone deacetylase) inhibitors on smHDAC8. Besides several active hydroxamates, we identified a thiol-based inhibitor that inhibited smHDAC8 activity in the micromolar range with unexpected selectivity over the human isotype, which has not been observed so far. The crystal structure of smHDAC8 complexed with the thiol derivative revealed that the inhibitor is accommodated in the catalytic pocket, where it interacts with both the catalytic zinc ion and the essential catalytic tyrosine (Y341) residue via its mercaptoacetamide warhead. To our knowledge, this is the first complex crystal structure of any HDAC inhibited by a mercaptoacetamide inhibitor, and therefore, this finding offers a rationale for further improvement. Finally, an ester prodrug of the thiol HDAC inhibitor exhibited antiparasitic activity on cultured schistosomes in a dose-dependent manner.     

Rescue by chloramphenicol and rifampicin of UV-irradiated lon, exrA and recA mutants of Escherichia coli K12

The effect of temporary treatment with chloramphenicol or rifampicin on the survival of UV irradiated cells of selected Escherichia coli K12 radiation sensitive mutants was examined. Increased survival resulted for both exrA and recA mutants, and also for the unsuppressed lon mutant, but cells of the parent strain and the recB mutant were not rescued. This contrasts with our earlier finding that after exposure of the bacteria to γ-rays, chloramphenicol treatment rescued the exrA and lon mutants but not the recA mutant. We now report that an exrA recA double mutant was rescued by chlramphenicol after UV radiation, but not after anaerobic ionizing radiation. Inclusion of inhibitors of uvrA governed repair, caffeine and 8-methoxypsoralen (8-MOP), in the incubation medium containing chloramphenicol, did not reduce the rescue of the exrA or recA mutants, although caffeine eliminated rescue of the lon mutant, which was itself unaffected by 8-MOP. However it is concluded that chlormaphenicol rescue of the exrA and recA mutants after UV radiation was not entirely independent of the excision-repair process, since the uvrA recA and uvrA exrA double mutants were not rescued by this treatment.     

8-Oxo-erythraline,a naturally-occurring principal alkaloid from Erythrina crista-galli

The principal alkaloid of the leaves of a commercially available variety of Erythrina crista-galli, has been identified by mass and ¹H NMR spectroscopy as 8-oxo-erythraline.     

Diterpenoids from Acacia leucophloea

From the root bark of Acacia leucophloea (Mimosaceae) two new pimar-8(14)-ene diterpenoids have been isolated. Their structures have been established by chemical and spectroscopic means as 1β,15R,16-trihydroxypimar8(14)-ene(leucophleol) and 15R,16-epoxy-1β,11α-dihydroxypimar-8(14)-ene(leucophleoxol).     

Novel 2, 8-bit derivatives of quinolines attenuate Pseudomonas aeruginosa virulence and biofilm formation

Signal molecules are stimulators of multiple quroum-sensing virulence and biofilm formation. Small molecule analogues have been suspected as a potent inhibitor in therapeutic strategy. Herein, we synthesized a series of small molecule compounds from the 2, 8-bit derivatives of quinoline by Suzuki coupling reaction. We found that these compounds have the biofilm inhibitory effect in normal condition instead of phosphate limitation state. Furthermore, lacZ reporter strain assay and rhamnolipids as well as pyocyanin experiments showed that these compounds did not affect las and pqs system but reduced the expression of rhl. All these results suggest that quinoline derivatives can be treated as potent inhibitors against biofilm and reduce virulence through the rhl system. This research will be useful in designing new quorum sensing inhibitors to attenuate the infection of bacteria.     

Optimization of 8-oxoadenines with toll-like-receptor 7 and 8 activity

Toll-like receptors 7 and 8 (TLR7/8) agonists are potent immunostimulants that are attracting considerable interest as vaccine adjuvants. We recently reported the synthesis of a new series of 2-O-butyl-8-oxoadenines substituted at the 9-position with various linkers and N-heterocycles, and showed that TLR7/8 selectivity, potency and cytokine induction could be modulated by varying the alkyl linker length and the N-heterocyclic ring. In the present study, we further optimized the oxoadenine scaffold by investigating the effect of different substituents at the 2-position of the oxoadenine on TLR7/8 potency/selectivity, cytokine induction and DC maturation in human PBMCs. The results show that introducing a 1-(S)-methylbutoxy group at the 2-position of the oxoadenine significantly increased potency for TLR7/8 activity, cytokine induction and DC maturation.     

Critical Role of 7,8-Didemethyl-8-hydroxy-5-deazariboflavin for Photoreactivation in Chlamydomonas reinhardtii

DNA photolyases use two noncovalently bound chromophores to catalyze photoreactivation, the blue light-dependent repair of DNA that has been damaged by ultraviolet light. FAD is the catalytic chromophore for all photolyases and is essential for photoreactivation. The identity of the second chromophore is often 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO). Under standard light conditions, the second chromophore is considered nonessential for photoreactivation because DNA photolyase bound to only FAD is sufficient to catalyze the repair of UV-damaged DNA. phr1 is a photoreactivation-deficient strain of Chlamydomonas. In this work, the PHR1 gene of Chlamydomonas was cloned through molecular mapping and shown to encode a protein similar to known FO synthases. Additional results revealed that the phr1 strain was deficient in an FO-like molecule and that this deficiency, as well as the phr1 photoreactivation deficiency, could be rescued by transformation with DNA constructs containing the PHR1 gene. Furthermore, expression of a PHR1 cDNA in Escherichia coli produced a protein that generated a molecule with characteristics similar to FO. Together, these results indicate that the Chlamydomonas PHR1 gene encodes an FO synthase and that optimal photoreactivation in Chlamydomonas requires FO, a molecule known to serve as a second chromophore for DNA photolyases.     

Low-dimensional compounds containing bioactive ligands. Part XIV: High selective antiproliferative activity of tris(5-chloro-8-quinolinolato)gallium(III) complex against human cancer cell lines

Four gallium(III) complexes, [Ga(ClQ)₃]⋅MeOH (1 – MeOH), [Ga(ClQ)₃] (1), [Ga(BrQ)₃] (2), [Ga(dIQ)₃] (3) and [Ga(CQ)₃] (4), were prepared (H-ClQ = 5-chloro-8-quinolinol, H-BrQ = 7-bromo-8-quinolinol, H-dIQ = 5,7-diiodo-8-quinolinol, H-CQ = 5-chloro-7-iodo-8-quinolinol) and characterised by elemental analysis, IR and NMR spectroscopy. Single crystal structure analysis of 1 – MeOH confirmed that the complex has a molecular structure with gallium(III) metal ion coordinated in mer-fashion by N- and O-donor atoms of three ClQ ligands. Stability of all complexes in DMSO was proved by ¹H NMR spectroscopy. The in vitro antiproliferative activity of 1 was evaluated against the A2780, MBA-MB-231 and HCT116 cell lines. Complex 1 displays higher antiproliferative activity (IC₅₀ values in the range 2.1–6 μm) compared to the ClQ ligand and cisplatin; and a significant selective antiproliferative potency (IC₅₀ = 136 μm, for normal MRC5pd30 cell line). Radical scavenging experiments revealed that complex 1 exhibits the highest antioxidant activity of the prepared complexes as well as the ligands.     

In vitro activities of a new fluoroquinolone derivative highly active against Chlamydia trachomatis

Chlamydia trachomatis is a bacterial human pathogen responsible for the development of trachoma, an infection leading to blindness, and is also the cause of the main bacterial sexually transmitted infection worldwide. We designed a new inhibitor of this bacterium with, however, some prerequisites using (i) the iron dependency of the bacterium, (ii) a commercially available broad-spectrum antibiotic and (iii) a short synthetic pathway. The corresponding 8-hydroxyquinoline-ciprofloxacin conjugate was evaluated against a panel of pathogenic bacteria, including C. trachomatis but also the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species). Its anti-Chlamydia activity is higher than that of ciprofloxacin and seems to be related to the fluoroquinolone moiety of the molecule, which is also responsible for the complexation of iron(III), as demonstrated by spectrophotometric titration.     

Different Cre systems induce differential microRNA landscapes and abnormalities in the female reproductive tracts of Dgcr8 conditional knockout mice

ObjectivesThe female reproductive tract comprises several different cell types. Using three representative Cre systems, we comparatively analysed the phenotypes of Dgcr8 conditional knockout (cKO) mice to understand the function of Dgcr8, involved in canonical microRNA biogenesis, in the female reproductive tract.Materials and Methods Dgcr8 ^f/f mice were crossed with Ltf ^icre/+, Amhr2 ^cre/+ or PR ^cre/+ mice to produce mice deficient in Dgcr8 in epithelial (Dgcr8 ^ed/ed), mesenchymal (Dgcr8 ^md/md) and all the compartments (Dgcr8 ^td/td) in the female reproductive tract. Reproductive phenotypes were evaluated in Dgcr8 cKO mice. Uteri and/or oviducts were used for small RNA‐seq, mRNA‐seq, real‐time RT‐PCR, and/or morphologic and histological analyses.Result Dgcr8 ^ed/ed mice did not exhibit any distinct defects, whereas Dgcr8 ^md/md mice showed sub‐fertility and oviductal smooth muscle deformities. Dgcr8 ^td/td mice were infertile due to anovulation and acute inflammation in the female reproductive tract and suffered from an atrophic uterus with myometrial defects. The microRNAs and mRNAs related to immune modulation and/or smooth muscle growth were systemically altered in the Dgcr8 ^td/td uterus. Expression profiles of dysregulated microRNAs and mRNAs in the Dgcr8 ^td/td uterus were different from those in other genotypes in a Cre‐dependent manner.Conclusions Dgcr8 deficiency with different Cre systems induces overlapping but distinct phenotypes as well as the profiles of microRNAs and their target mRNAs in the female reproductive tract, suggesting the importance of selecting the appropriate Cre driver to investigate the genes of interest.     

Chirality and biosynthesis of lilac compounds in Actinidia arguta flowers

Biosynthesis of lilac compounds in ‘Hortgem Tahi’ kiwifruit (Actinidia arguta) flowers was investigated by treating inflorescences with d₅-linalool. The incorporation of the deuterium label into 8-hydroxylinalool, 8-oxolinalool, the lilac aldehydes, alcohols, and alcohol epoxides was followed by GC-MS and enantioselective GC-MS. Both (R)- and (S)-linalool were produced naturally by the flowers, but 8-hydroxylinalool, 8-oxolinalool, and the lilac aldehydes and alcohols occurred predominantly as the (S) and 5′(S)-diastereoisomers, respectively. The enantioselective step in the biosynthesis of the lilac aldehydes and alcohols was concluded to be the oxidation of linalool to (S)-8-hydroxylinalool. In contrast, the lilac alcohol epoxides had a 5′(R):(S) ratio, the same as for linalool, which suggests that either these compounds are not synthesised from the 5′(S)-configured lilac aldehydes and alcohols, or that if indeed they are, then it is by an enantioselective step that favours utilisation of the 5′(R)-configured compounds.     

Inactivation of a common OGG1 variant by TNF-alpha in mammalian cells

Reactive oxygen species threaten genomic integrity by inducing oxidative DNA damage. One common form of oxidative DNA damage is the mutagenic lesion 8-oxoguanine (8-oxodG). One driver of oxidative stress that can induce 8-oxodG is inflammation, which can be initiated by the cytokine tumor necrosis factor alpha (TNF-α). Oxidative DNA damage is primarily repaired by the base excision repair pathway, initiated by glycosylases targeting specific DNA lesions. 8-oxodG is excised by 8-oxoguanine glycosylase 1 (OGG1). A common Ogg1 allelic variant is S326C-Ogg1, prevalent in Asian and Caucasian populations. S326C-Ogg1 is associated with various forms of cancer, and is inactivated by oxidation. However, whether oxidative stress caused by inflammatory cytokines compromises OGG1 variant repair activity remains unknown. We addressed whether TNF-α causes oxidative stress that both induces DNA damage and inactivates S326C-OGG1 via cysteine 326 oxidation. In mouse embryonic fibroblasts, we found that S326C-OGG1 was inactivated only after exposure to H₂O₂ or TNF-α. Treatment with the antioxidant N-acetylcysteine prior to oxidative stress rescued S326C-OGG1 activity, demonstrated by in vitro and cellular repair assays. In contrast, S326C-OGG1 activity was unaffected by potassium bromate, which induces oxidative DNA damage without causing oxidative stress, and presumably cysteine oxidation. This study reveals that Cys326 is vulnerable to oxidation that inactivates S326C-OGG1. Physiologically relevant levels of TNF-α simultaneously induce 8-oxodG and inactivate S326C-OGG1. These results suggest a mechanism that could contribute to increased risk of cancer among S326C-Ogg1 homozygous individuals.