Selective vitamin D receptor modulators and their effects on colorectal tumor growth

The active form of vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], is an endocrine hormone whose classic role is the maintenance of calcium homeostasis. It is well documented that 1,25(OH)(2)D(3) also has anti-tumor effects on a number of cancers and cancer cell lines including breast, colorectal, gastric, liver, ovarian, prostate, and non-melanoma skin cancers. Included in the anti-tumor activities of 1,25(OH)(2)D(3) are its ability to cause antiproliferation, prodifferentation and decrease angiogenesis. Furthermore, through regulation of the plaminogen activator (PA) system and a class of proteolytic enzymes called matrix metalloproteinases (MMPs), 1,25(OH)(2)D(3) reduces the invasive spread of tumor cells. Because of the calcemic limitations of using 1,25(OH)(2)D(3) as a therapy, we have tested the effects of a novel Gemini vitamin D analogue, Deuterated Gemini (DG), on mouse colorectal cancer. We demonstrated that DG is more potent in reducing tumor volume and mass, compared to control and 1,25(OH)(2)D(3). DG significantly prevented (100% reduction, p<0.05) the invasive spread of colorectal tumor cells into the surrounding muscle, and had no effect on serum calcium levels. Thus, DG acts as a selective vitamin D receptor modulator (SVDRM) by enhancing select anti-tumor characteristic 1,25(OH)(2)D(3) activities, without inducing hypercalcemia. Thus, DG shows promise in the development of colorectal cancer therapies.     

Priming by airborne signals boosts direct and indirect resistance in maize

Plants counteract attack by herbivorous insects using a variety of inducible defence mechanisms. The production of toxic proteins and metabolites that instantly affect the herbivore's development are examples of direct induced defence. In addition, plants may release mixtures of volatile organic compounds (VOCs) that indirectly protect the plant by attracting natural enemies of the herbivore. Recent studies suggest that these VOCs can also prime nearby plants for enhanced induction of defence upon future insect attack. However, evidence that this defence priming causes reduced vulnerability to insects is sparse. Here we present molecular, chemical and behavioural evidence that VOC-induced priming leads to improved direct and indirect resistance in maize. A differential hybridization screen for inducible genes upon attack by Spodoptera littoralis caterpillars identified 10 defence-related genes that are responsive to wounding, jasmonic acid (JA), or caterpillar regurgitant. Exposure to VOCs from caterpillar-infested plants did not activate these genes directly, but primed a subset of them for earlier and/or stronger induction upon subsequent defence elicitation. This priming for defence-related gene expression correlated with reduced caterpillar feeding and development. Furthermore, exposure to caterpillar-induced VOCs primed for enhanced emissions of aromatic and terpenoid compounds. At the peak of this VOC emission, primed plants were significantly more attractive to parasitic Cotesia marginiventris waSPS. This study shows that VOC-induced priming targets a specific subset of JA-inducible genes, and links these responses at the molecular level to enhanced levels of direct and indirect resistance against insect attack.     

Selector Screening for Enantioseparation of dl‐α‐Methyl Phenylglycine Amide by Liquid–Liquid Extraction

Enantioseparation through liquid extraction technology is an emerging field, e.g., enantioseparations of amino acids (and derivatives thereof), amino alcohols, amines, and carboxylic acids have been reported. Often, when a new selector is developed, the versatility of substrate scope is investigated. From an industrial point of view, the problem is typically approached the other way around, and for a target racemate, a selector needs to be found in order to accomplish the desired enantioseparation. This study presents such a screening approach for the separation of the enantiomers of dl ‐α‐methyl phenylglycine amide (dl ‐α‐MPGA), a model amide racemate with high industrial relevance. Chiral selectors that were reported for other classes of racemates were investigated, i.e., several macrocyclic selectors and Pd‐BINAP complexes. It appeared very challenging to obtain both high extraction yields and good enantioselectivity for most selectors, but Pd‐BINAP‐based selectors performed well, with enantioselectivities up to 7.4 with an extraction yield of the desired enantiomer of 95.8%. These high enantioselectivities were obtained using dichloromethane as solvent. Using less volatile chlorobenzene or 1‐chloropentane, reasonable selectivities of up to 1.7 were measured, making these the best alternative solvents for dichloromethane. Chirality 27:123–130, 2015. © 2014 Wiley Periodicals, Inc.     

A thiol‐disulfide oxidoreductase of the Gram‐positive pathogen Corynebacterium diphtheriae is essential for viability,pilus assembly,toxin production and virulence

The Gram‐positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include the key virulence factors diphtheria toxin and the adhesive pili. How these proteins attain their native conformations after translocation as unfolded precursors remains elusive. The fact that the majority of these exported proteins contain multiple cysteine residues and that several membrane‐bound oxidoreductases are encoded in the corynebacterial genome suggests the existence of an oxidative protein‐folding pathway in this organism. Here we show that the shaft pilin SpaA harbors a disulfide bond in vivo and alanine substitution of these cysteines abrogates SpaA polymerization and leads to the secretion of degraded SpaA peptides. We then identified a thiol‐disulfide oxidoreductase (MdbA), whose structure exhibits a conserved thioredoxin‐like domain with a CPHC active site. Remarkably, deletion of mdbA results in a severe temperature‐sensitive cell division phenotype. This mutant also fails to assemble pilus structures and is greatly defective in toxin production. Consistent with these defects, the ΔmdbA mutant is attenuated in a guinea pig model of diphtheritic toxemia. Given its diverse cellular functions in cell division, pilus assembly and toxin production, we propose that MdbA is a component of the general oxidative folding machine in C. diphtheriae.     

Durable broad-spectrum powdery mildew resistance in pea er1 plants is conferred by natural loss-of-function mutations in PsMLO1

Loss-of-function alleles of plant-specific MLO (Mildew Resistance Locus O) genes confer broad-spectrum powdery mildew resistance in monocot (barley) and dicot (Arabidopsis thaliana, tomato) plants. Recessively inherited powdery mildew resistance in pea (Pisum sativum) er1 plants is, in many aspects, reminiscent of mlo-conditioned powdery mildew immunity, yet the underlying gene has remained elusive to date. We used a polymerase chain reaction (PCR)-based approach to amplify a candidate MLO cDNA from wild-type (Er1) pea. Sequence analysis of the PsMLO1 candidate gene in two natural er1 accessions from Asia and two er1-containing pea cultivars with a New World origin revealed, in each case, detrimental nucleotide polymorphisms in PsMLO1, suggesting that PsMLO1 is Er1. We corroborated this hypothesis by restoration of susceptibility on transient expression of PsMLO1 in the leaves of two resistant er1 accessions. Orthologous legume MLO genes from Medicago truncatula and Lotus japonicus likewise complemented the er1 phenotype. All tested er1 genotypes showed unaltered colonization with the arbuscular mycorrhizal fungus, Glomus intraradices, and with nitrogen-fixing rhizobial bacteria. Our data demonstrate that PsMLO1 is Er1 and that the loss of PsMLO1 function conditions durable broad-spectrum powdery mildew resistance in pea.     

Effect of CD44 deficiency on in vitro and in vivo osteoclast formation

In vitro studies have shown that CD44 is involved in the fusion process of osteoclast precursor cells. Yet, in vivo studies do not support this, since an osteopetrotic phenotype has not been described for CD44 knock-out (CD44 k.o.) mice. This discrepancy may suggest that the role of CD44 in fusion may depend on the microenvironment of osteoclast formation. We investigated osteoclast formation of CD44 k.o. and wild-type mice under three conditions: in vitro, both on plastic and on bone and in vivo by analyzing osteoclast number, and size in long bones from wild-type and CD44 k.o. mice. Bone marrow cells from wild-type and CD44 k.o. mice were analyzed for their capacity to form osteoclasts on plastic and on bone in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). On plastic, the number of multinucleated tartrate resistant acid phosphatase (TRAP) positive cells in CD44 k.o. cultures was twofold higher than in wild-type cultures. On bone, however, equal numbers of osteoclasts were formed. Interestingly, the total number of osteoclasts formed on bone proved to be higher than on plastic for both genotypes, strongly suggesting that osteoclastogenesis was stimulated by the bone surface, and that CD44 is not required for osteoclast formation on bone. Functional analyses showed that bone resorption was similar for both genotypes. We further studied the osteoclastogenic potential of wild-type bone marrow cells in the presence of CD44 blocking antibodies. Osteoclastogenesis was not affected by these antibodies, a further indication that CD44 is not required for the formation of multinucleated cells. Finally, we analyzed the in vivo formation of osteoclasts by analyzing long bones from wild-type and CD44 k.o. mice. Morphometric analysis revealed no difference in osteoclast number, nor in number of nuclei per osteoclasts or in osteoclast size. Our in vitro experiments on plastic showed an enhanced formation of osteoclasts in the absence of CD44, thus suggesting that CD44 has an inhibitory effect on osteoclastogenesis. However, when osteoclasts were generated on bone, no differences in number of multinucleated cells nor in bone resorption were seen. These observations are in agreement with in vivo osteoclast characteristics, where no differences between wild-type and CD44 k.o. bones were encountered. Therefore, the modulating role of CD44 in osteoclast formation appears to depend on the microenvironment.     

The first enantioselective synthesis of the amavadin ligand and its complexation to vanadium

The ligand of the naturally occurring vanadium compound amavadin found in Amanita muscaria, (2S, 2'S)-N-hydroxyimino-2,2'-dipropionic acid (1), was synthesized stereoselectively in two steps with 43% overall yield. After complexation of this ligand to vanadyl acetate, amavadin was isolated in quantitative yield. Due to the chirality at vanadium amavadin consists of a mixture of delta and lambda diastereoisomers. Directly after its synthesis, the delta to lambda ratio of amavadin is 2.27 and it decreases to 0.80 after equilibrium has been reached. During this epimerization the optical rotation for V[(2S,2'S)-N-hydroxyimino-(2,2')-dipropionate]2 (=amavadin) changes from [alpha](D)25 = +36 degrees to +114.0 degrees (c = 0.5, H2O). For V[(2R,2'R)-N-hydroxyimino-(2,2')-dipropionate] the optical rotation changes from [alpha](D)25 = -36 degrees to -113.2 degrees (c = 0.5, H2O).     

Large complex formation of the inhibitor of caspase-activated DNase

Inhibitor of caspase-activated DNase (ICAD) is required for correctly folding of CAD and inhibits nuclease activity of CAD in non-apoptotic cells. From proteomic analysis of the ICAD binding proteins, we revealed that over-expressed flag-ICAD bound other ICAD molecules}. Purified recombinant ICAD protein showed three bands, 66 KDa, 132 KDa and 450 KDa, by native-PAGE. ICAD fused with glutathione-S-transferase (GST) was immunoprecipitated with anti-flag antibody from Jurkat cell lysates cotransfected with ICAD fused with either GST or flag expression vectors. When purified recombinant ICAD protein was separated by gel chromatography, the molecular weight of ICAD was detected at 440 and 45 K. ICAD in extracts of wild type Jurkat cells also existed at 440 and 45 K as measured by gel chromatography; so that fractions of CAD coincided with fractions of 440 K of ICAD. These results indicate that ICAD and/or CAD appeared to form large complexes in Jurkat cells.     

Nuclear transfer of perchloric acid-soluble protein by endoplasmic reticulum stressors

Perchloric acid-soluble protein (PSP) is highly conserved during evolution from bacteria to mammals. Although PSP has been recognized as an inhibitor of translation and proliferation in vitro, its precise biological role has not yet been elucidated. Since we previously found similar distributions for PSP and the endoplasmic reticulum (ER) and Golgi complex, the intracellular distribution of PSP was analyzed in more detail. Immunofluorescence studies indicated that PSP co-localized with the ER and Golgi complex, since the distribution pattern of PSP was well matched to both of these organelles. An immunoelectron microscopic study revealed PSP was located not only in the cytosol but also on the surface of the outer ER membrane. Since PSP was present on the ER, we speculated that it may be associated with ER function. Therefore, we analyzed whether or not the ER stress response, which is one of the ER functions, affected PSP expression. The results showed that various ER stressors (thapsigargin, A23187, tunicamycin, brefeldin A, and cisplatin) provoked a dramatic change in the localization of PSP from outside of the nucleus to inside the nucleus within 3 h. Moreover, the ER stressors induced PSP expression. These results suggest that PSP is involved in the cellular response to ER stressors, and that the change in localization of PSP from the ER to the nucleus may be associated with ER stress responses.     

Nucleotide-binding oligomerization domain-2 modulates specific TLR pathways for the induction of cytokine release

The recognition of peptidoglycan by cells of the innate immune system has been controversial; both TLR2 and nucleotide-binding oligomerization domain-2 (NOD2) have been implicated in this process. In the present study we demonstrate that although NOD2 is required for recognition of peptidoglycan, this leads to strong synergistic effects on TLR2-mediated production of both pro- and anti-inflammatory cytokines. Defective IL-10 production in patients with Crohn's disease bearing loss of function mutations of NOD2 may lead to overwhelming inflammation due to a subsequent Th1 bias. In addition to the potentiation of TLR2 effects, NOD2 is a modulator of signals transmitted through TLR4 and TLR3, but not through TLR5, TLR9, or TLR7. Thus, interaction between NOD2 and specific TLR pathways may represent an important modulatory mechanism of innate immune responses.