Disruption of Nucleotide Homeostasis by the Antiproliferative Drug 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside Monophosphate (AICAR)

5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside monophosphate (AICAR) is a natural metabolite with potent anti-proliferative and low energy mimetic properties. At high concentration, AICAR is toxic for yeast and mammalian cells, but the molecular basis of this toxicity is poorly understood. Here, we report the identification of yeast purine salvage pathway mutants that are synthetically lethal with AICAR accumulation. Genetic suppression revealed that this synthetic lethality is in part due to low expression of adenine phosphoribosyl transferase under high AICAR conditions. In addition, metabolite profiling points to the AICAR/NTP balance as crucial for optimal utilization of glucose as a carbon source. Indeed, we found that AICAR toxicity in yeast and human cells is alleviated when glucose is replaced by an alternative carbon source. Together, our metabolic analyses unveil the AICAR/NTP balance as a major factor of AICAR antiproliferative effects.     

Functional Expression of P-Glycoprotein in an Immortalised Cell Line of Rat Brain Endothelial Cells, RBE4

Abstract: The presence of P-glycoprotein in the cell plasma membrane limits the penetration of many cytotoxic substances into cells that express the gene product. There is considerable evidence also to indicate that P-glycoprotein is expressed as part of the normal blood-brain barrier in the luminal membranes of the cerebral capillary endothelial cells, where it presumably performs a protective function for the brain. This report describes the functional expression of P-glycoprotein in an immortalised cell line, RBE4, derived from rat cerebral capillary endothelial cells. The expression of P-glycoprotein is demonstrated by western immunoblotting and by immunogold and fluorescent staining with monoclonal antibodies. The cellular accumulation of [³H]colchicine and [³H]vinblastine is investigated and shown to be enhanced by the presence of azidothymidine, chlorpromazine, verapamil, cyclosporin A, and PSC 833 ([3'-keto-Bmt¹]-[Val²]-cyclosporin) at 50 or 100 µ M concentration. It is concluded that the RBE4 cell line is a valuable tool for investigating the mechanisms of P-glycoprotein activity both in the blood-brain barrier and in multidrug resistance in general.     

CpG Methylation Changes within the IL2RA Promoter in Type 1 Diabetes of Childhood Onset

None of the polymorphic variants of the IL2RA gene found associated with Type 1 Diabetes (T1D) was shown to have a functional effect. To test if the epigenetic variation could play a role at this locus, we studied the methylation of 6 CpGs located within the proximal promoter of IL2RA gene in 252 T1D patients compared with 286 age-matched controls. We found that DNA methylation at CpGs −373 and −456 was slightly but significantly higher in patients than in controls (40.4±4.6 vs 38.3±5.4, p = 1.4E4; 91.4±2.8 vs 89.5±5.3, p = 1.8E-6), while other CpG showed a strictly comparable methylation. Among 106 single nucleotide polymorphisms (SNPs) located in the neighboring 180kb region, we found that 28 SNPs were associated with DNA methylation at CpG −373. Sixteen of these SNPs were known to be associated with T1D. Our findings suggest that the effect of IL2RA risk alleles on T1D may be partially mediated through epigenetic changes.     

Novel lipophilic 7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid derivatives as potential antitumor agents: improved synthesis and in vitro evaluation

A convenient route for the synthesis of some acyloxymethyl esters and carboxamides of levofloxacin (LV) with modulated lipophilicity is described. The synthesized compounds were evaluated in vitro for their growth inhibitory effect in five human cancer cell lines. The most efficient LV derivatives (ester 2e and amide 4d) displayed IC(50) values in the 0.2-2.2 μM range, while IC(50) values for parent LV ranged between 70 and 622 μM depending on the cell line. The esters displayed no in vivo toxicity up to 80 mg/kg when administered intraperitoneally. This study thus shows that LV analogs displayed antitumor efficacy, at least in vitro, a feature that appeared to be independent from the lipophilicity of the grafted substituent.     

Crop planting dates: an analysis of global patterns

Aim To assemble a data set of global crop planting and harvesting dates for 19 major crops, explore spatial relationships between planting date and climate for two of them, and compare our analysis with a review of the literature on factors that drive decisions on planting dates. Location Global. Methods We digitized and georeferenced existing data on crop planting and harvesting dates from six sources. We then examined relationships between planting dates and temperature, precipitation and potential evapotranspiration using 30‐year average climatologies from the Climatic Research Unit, University of East Anglia (CRU CL 2.0). Results We present global planting date patterns for maize, spring wheat and winter wheat (our full, publicly available data set contains planting and harvesting dates for 19 major crops). Maize planting in the northern mid‐latitudes generally occurs in April and May. Daily average air temperatures are usually c. 12–17 °C at the time of maize planting in these regions, although soil moisture often determines planting date more directly than does temperature. Maize planting dates vary more widely in tropical regions. Spring wheat is usually planted at cooler temperatures than maize, between c. 8 and 14 °C in temperate regions. Winter wheat is generally planted in September and October in the northern mid‐latitudes. Main conclusions In temperate regions, spatial patterns of maize and spring wheat planting dates can be predicted reasonably well by assuming a fixed temperature at planting. However, planting dates in lower latitudes and planting dates of winter wheat are more difficult to predict from climate alone. In part this is because planting dates may be chosen to ensure a favourable climate during a critical growth stage, such as flowering, rather than to ensure an optimal climate early in the crop's growth. The lack of predictability is also due to the pervasive influence of technological and socio‐economic factors on planting dates.     

Maturation stress generation in poplar tension wood studied by synchrotron radiation microdiffraction

Tension wood is widespread in the organs of woody plants. During its formation, it generates a large tensile mechanical stress called maturation stress. Maturation stress performs essential biomechanical functions such as optimizing the mechanical resistance of the stem, performing adaptive movements, and ensuring the long-term stability of growing plants. Although various hypotheses have recently been proposed, the mechanism generating maturation stress is not yet fully understood. In order to discriminate between these hypotheses, we investigated structural changes in cellulose microfibrils along sequences of xylem cell differentiation in tension and normal wood of poplar (Populus deltoides × Populus trichocarpa 'I45-51'). Synchrotron radiation microdiffraction was used to measure the evolution of the angle and lattice spacing of crystalline cellulose associated with the deposition of successive cell wall layers. Profiles of normal and tension wood were very similar in early development stages corresponding to the formation of the S1 layer and the outer part of the S2 layer. Subsequent layers were found with a lower microfibril angle (MFA), corresponding to the inner part of the S2 layer of normal wood (MFA approximately 10°) and the G layer of tension wood (MFA approximately 0°). In tension wood only, this steep decrease in MFA occurred together with an increase in cellulose lattice spacing. The relative increase in lattice spacing was found close to the usual value of maturation strains. Analysis showed that this increase in lattice spacing is at least partly due to mechanical stress induced in cellulose microfibrils soon after their deposition, suggesting that the G layer directly generates and supports the tensile maturation stress in poplar tension wood.     

Synchrotron ultraviolet microspectroscopy on rat cortical bone: involvement of tyrosine and tryptophan in the osteocyte and its environment

Alcohol induced osteoporosis is characterized by a bone mass decrease and microarchitecture alterations. Having observed an excess in osteocyte apoptosis, we aimed to assess the bone tissue biochemistry, particularly in the osteocyte and its environment. For this purpose, we used a model of alcohol induced osteoporosis in rats. Bone sections of cortical bone were investigated using synchrotron UV-microspectrofluorescence at subcellular resolution. We show that bone present three fluorescence peaks at 305, 333 and 385 nm, respectively corresponding to tyrosine, tryptophan and collagen. We have determined that tyrosine/collagen and tryptophan/collagen ratios were higher in the strong alcohol consumption group. Tryptophan is related to the serotonin metabolism involved in bone formation, while tyrosine is involved in the activity of tyrosine kinases and phosphatases in osteocytes. Our experiment represents the first combined synchrotron UV microspectroscopy analysis of bone tissue with a quantitative biochemical characterization in the osteocyte and surrounding matrix performed separately.     

Uterine contractions depend on KIT-positive interstitial cells in the mouse: genetic and pharmacological evidence

In the gastrointestinal tract, interstitial cells of Cajal (ICCs) generate a pacemaker activity. They produce electric slow waves that trigger and coordinate gut smooth muscle contractions. Interstitial cells of Cajal's slender shape is revealed by KIT immunostaining. Based on several features, including KIT expression and KIT dependence, ICC-like cells were identified in nongastrointestinal tissues. Here, we investigated in the mouse whether uterine contractions depend on ICC-like cells' activity. By labeling KIT-expressing cells, we found putative ICC-like cells in the uterus, observed as KIT-positive interstitial, long spindle-shaped cells with fine branched cytoplasm processes, distributed in muscular layers and in subepithelial connective tissue. We then checked the potential KIT dependence of ex vivo contractile activity of the uterus by combining genetic and pharmacological approaches, using the Kit W-v hypomorphic mutation, and imatinib as a KIT noncompetitive inhibitor. We found a significant reduction in frequency of longitudinal uterine contractions in Kit W-v/Kit W-v compared with Kit+/+ mice, whereas amplitude was unaffected. There was no difference in frequency or amplitude of circular uterine contractions between Kit W-v/Kit W-v and Kit+/+ mice. Ex vivo treatment of Kit+/+ uterine horns with imatinib resulted in a dose-dependent reduction of the frequency and amplitude of longitudinal myometrial contractions. Amplitude and frequency of circular contractions were unaffected in presence of imatinib. These concurrent results suggest that longitudinal contractions of the uterus depend on a KIT signaling pathway of ICC-like cells. The existence of ICC-like cells in the myometrium may enhance our understanding of uterine spontaneous contractile activity and suggest new approaches for treatment of uterine contractility disorders.