Synthesis of α-carboxyphosphinopeptides derived from norleucine

In the present study, we describe in detail the synthesis of a relatively rare class of phosphorus compounds, α-carboxyphosphinopeptides. We prepared several norleucine-derived α-carboxyphosphinic pseudopeptides of the general formula Nle-Ψ[PO(OH)]-Gly. These compounds could have important applications as transition state-mimicking inhibitors for methionine or leucine aminopeptidases or other enzymes. For the preparation of the key α-carboxyphosphinate protected precursors, we investigated, compared and improved two different synthetic methods described in literature: the Arbuzov reaction of a silylated N-protected phosphinic acid with a bromoacetate ester and the nucleophilic addition of a mixed O-methyl S-phenyl N-protected phosphonic acid or a methyl N-protected phosphonochloridate with tert-butyl lithioacetate. We also prepared two N-Fmoc protected synthons, Fmoc-Nle-Ψ[PO(OH)]-Gly-COOH and Fmoc-Nle-Ψ[PO(OAd)]-Gly-COOH, and demonstrated that these precursors are suitable building blocks for the solid-phase synthesis of α-carboxyphosphinopeptides.     

Hematological,biochemical, respiratory,cardiovascular and electroneurophysiological parameters in African green monkeys (Cercopithecus aethiops sabaeus). Its use in non‐clinical toxicological studies

Background The purpose of this study is to better characterize the hematological, biochemical, respiratory, cardiovascular and electroneurophysiological parameters in young adult Cercopithecus aethiops sabaeus of both sexes. The rhesus and cynomolgus monkeys are widely used as experimental primate models. However, only few articles have been published testing toxicological effects of pharmaceuticals on African green monkey. Methods The present study was carried out with the recompilation of all parameters recorded before the first drug administration in five sub‐chronic or chronic toxicological studies performed on 66 Cercopithecus aethiops sabaeus, born in Cuba. Results This study provides hematological, biochemical, respiratory, cardiovascular and electroneurophysiological data for both choosing animals to be included into experiments and monitoring these parameters during the study. Conclusions We conclude that this study provides valuable integrated data for determining the health status, including electroneurophysiological parameters, data not previously reported for this species, of the African green monkey.     

Association between TAS2R38 gene polymorphisms and colorectal cancer risk: a case-control study in two independent populations of Caucasian origin

Molecular sensing in the lingual mucosa and in the gastro-intestinal tract play a role in the detection of ingested harmful drugs and toxins. Therefore, genetic polymorphisms affecting the capability of initiating these responses may be critical for the subsequent efficiency of avoiding and/or eliminating possible threats to the organism. By using a tagging approach in the region of Taste Receptor 2R38 (TAS2R38) gene, we investigated all the common genetic variation of this gene region in relation to colorectal cancer risk with a case-control study in a German population (709 controls and 602 cases) and in a Czech population (623 controls and 601 cases). We found that there were no significant associations between individual SNPs of the TAS2R38 gene and colorectal cancer in the Czech or in the German population, nor in the joint analysis. However, when we analyzed the diplotypes and the phenotypes we found that the non-taster group had an increased risk of colorectal cancer in comparison to the taster group. This association was borderline significant in the Czech population, (OR = 1.28, 95% CI 0.99–1.67; P_value = 0.058) and statistically significant in the German population (OR = 1.36, 95% CI 1.06–1.75; P_value = 0.016) and in the joint analysis (OR = 1.34, 95% CI 1.12–1.61; P_value = 0.001). In conclusion, we found a suggestive association between the human bitter tasting phenotype and the risk of CRC in two different populations of Caucasian origin.     

Analysis of epithelial and mesenchymal markers in ovarian cancer reveals phenotypic heterogeneity and plasticity

In our studies of ovarian cancer cells we have identified subpopulations of cells that are in a transitory E/M hybrid stage, i.e. cells that simultaneously express epithelial and mesenchymal markers. E/M cells are not homogenous but, in vitro and in vivo, contain subsets that can be distinguished based on a number of phenotypic features, including the subcellular localization of E-cadherin, and the expression levels of Tie2, CD133, and CD44. A cellular subset (E/M-MP) (membrane E-cadherin(low)/cytoplasmic E-cadherin(high)/CD133(high), CD44(high), Tie2(low)) is highly enriched for tumor-forming cells and displays features which are generally associated with cancer stem cells. Our data suggest that E/M-MP cells are able to differentiate into different lineages under certain conditions, and have the capacity for self-renewal, i.e. to maintain a subset of undifferentiated E/M-MP cells during differentiation. Trans-differentiation of E/M-MP cells into mesenchymal or epithelial cells is associated with a loss of stem cell markers and tumorigenicity. In vivo xenograft tumor growth is driven by E/M-MP cells, which give rise to epithelial ovarian cancer cells. In contrast, in vitro, we found that E/M-MP cells differentiate into mesenchymal cells, in a process that involves pathways associated with an epithelial-to-mesenchymal transition. We also detected phenotypic plasticity that was dependent on external factors such as stress created by starvation or contact with either epithelial or mesenchymal cells in co-cultures. Our study provides a better understanding of the phenotypic complexity of ovarian cancer and has implications for ovarian cancer therapy.     

Attraction of ambrosia and bark beetles to coast live oaks infected by Phytophthora ramorum

1 Sudden oak death is caused by the apparently introduced oomycete, Phytophthora ramorum. We investigated the role of bark and ambrosia beetles in disease progression in coast live oaks Quercus agrifolia. 2 In two Marin County, California sites, 80 trees were inoculated in July 2002 with P. ramorum and 40 were wounded without inoculation. Half of the trees in each group were sprayed with the insecticide permethrin [cyclopropanecarboxylic acid, 3‐(2,2‐dichloroethenyl)‐2,2‐dimethyl‐(3‐phenoxyphenyl) methyl ester] to prevent ambrosia and bark beetle attacks, and then were sprayed twice per year thereafter. After each treatment, sticky traps were placed on only the permethrin‐treated trees. Beetles were collected periodically in 2003. 3 Inoculated trees accounted for 95% of all beetles trapped. The ambrosia beetles Monarthrum scutellare and Xyleborinus saxeseni and the western oak bark beetle Pseudopityophthorus pubipennis were the most abundant of the seven species trapped. 4 Permethrin treatment delayed initiation of beetle attacks and significantly reduced the mean number of attacks per tree. Beetles did not attack any wounded or noncankered inoculated trees. 5 Trees with larger cankers trapped more beetles early in the disease. Once permethrin lost effectiveness, the number of beetle entrance tunnels was a more reliable predictor of subsequent trap catch than was canker size. 6 Beetles were initially attracted to P. ramorum cankers in response to kairomones generated in the host‐pathogen interaction. After beetles attacked the permethrin‐treated trees, aggregation pheromones most probably were the principal factor in beetle colonization behaviour.     

Biological Effects of Trichoderma harzianum Peptaibols on Mammalian Cells

Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 μg [dry weight] ml of extended boar semen⁻¹). The same exposure concentration depleted the boar sperm cells of NADH₂. Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C₁₈ cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Δψ_m) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C₁₈ cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS.     

The amitochondriate eukaryote Trichomonas vaginalis contains a divergent thioredoxin-linked peroxiredoxin antioxidant system

Trichomonas is an amitochondriate parasitic protozoon specialized for an anaerobic lifestyle. Nevertheless, it is exposed to oxygen and is able to cope with the resultant oxidative stress. In the absence of glutathione, cysteine has been thought to be the major antioxidant. We now report that the parasite contains thioredoxin reductase, which functions together with thioredoxin and thioredoxin peroxidase to detoxify potentially damaging oxidants. Thioredoxin reductase and thioredoxin also reduce cystine and so may play a role in maintaining the cellular cysteine levels. The importance of the thioredoxin system as one of the major antioxidant defense mechanisms in Trichomonas was confirmed by showing that the parasite responds to environmental changes resulting in increased oxidative stress by up-regulating thioredoxin and thioredoxin peroxidases levels. Sequence data indicate that the thioredoxin reductase of Trichomonas differs fundamentally in structure from that of its human host and thus may represent a useful drug target. The protein is generally similar to thioredoxin reductases present in other lower eukaryotes, all of which probably originated through horizontal gene transfer from a prokaryote. The phylogenetic signal in thioredoxin peroxidase is weak, but evidence from trees suggests that this gene has been subject to repeated horizontal gene transfers from different prokaryotes to different eukaryotes. The data are thus consistent with the complexity hypothesis that predicts that the evolution of simple pathways such as the thioredoxin cascade are likely to be affected by horizontal gene transfer between species.     

New steroidal saponins of Agave americana

Two new saponins, agavasaponin E and agavasaponin H have been isolated from the methanolic extract of Agave americana leaves and their structures elucidated. Agavasaponin E is 3-O-[β-d-xylopyranosyl-(1→2glc1)-α-l-rhamnopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→3glc 1)-β-d-glucopyranosyl-(1→4)-β-d-glucopyranosyl-(1→4)-α-d-galactopyranosyl]-(25R)-5α-spirostan-12-on-3β-ol, whereas agavasaponin H is 3-O-[β-d-xylopyranosyl-(1→2 glc 1)-α-l-rhamnopyranosyl-(1→4)-α-l-rhamnopyranosyl-(1→3 glc 1)-β-d-glucopyranosyl-(1→4)-β-d-glucopyranosyl-(1→4)-β-d-galactopyranosyl]-26-O-[β-d-glucopyranosyl]-(25R)-5α-furostan-12-on-3β,22α,26-triol.     

Sympatric diploid and hexaploid cytotypes of Senecio carniolicus (Asteraceae) in the Eastern Alps are separated along an altitudinal gradient

We explored the fine-scale distribution of cytotypes of the mountain plant Senecio carniolicus along an altitudinal transect in the Eastern Alps. Cytotypes showed a statistically significant altitudinal segregation with diploids exclusively found in the upper part of the transect, whereas diploids and hexaploids co-occurred in the lower range. Analysis of accompanying plant assemblages revealed significant differences between cytotypes along the entire transect but not within the lower part only, where both cytotypes co-occur. This suggests the presence of ecological differentiation between cytotypes with the diploid possessing the broader ecological niche. No tetraploids were detected, indicating the presence of strong crossing barriers. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.