SAR studies of 1,5-diarylpyrazole-based CCK1 receptor antagonists

A high throughput screening campaign revealed compound 1 as a potent antagonist of the human CCK(1) receptor. Here, we report the syntheses and SAR studies of 1,5-diarylpyrazole analogs with various structural modifications of the alkane side chain of the molecule. The difference in affinity between the two enantiomers for the CCK(1) receptor and the flexible nature of the linker led to the design of constrained analogs with increased potency.     

Metal binding of metallothioneins in human astrocytomas (U87 MG,IPDDC-2A)

Astroglia cells structurally and nutritionally support neurons in the central nervous system. They play an important role in guiding the construction of the nervous system and controlling the chemical and ionic environment of neurons. They also represent the major sites for accumulation and immobilisation of toxic metal ions most probably connected with metallothioneins. For this reason astroglia cells possess high cytosolic levels of metallothioneins I, II and III (MT-I,II,III). Our aim was to establish the inducibility and metal binding of MTs in two human astrocytoma cell lines, U87 MG (astrocytoma–glioblastoma, grade IV) and IPDDC-2A (astrocytoma, grade II), on exposure to cadmium chloride (1 μM). MTs were identified by molecular weight (size exclusion chromatography) and their metal content (Cd, Zn and Cu) to follow the interactions between metals. We showed that MTs are constitutively expressed in both human astrocytoma cell lines. In accordance with the higher malignancy grade of U87 MG, the amount of MTs was higher in U87 MG than in IPDDC-2A cells. After 24 hours of exposure to Cd their expression greatly increased in both cell lines and they were capable of immobilising almost all water soluble Cd. Induction of MTs in U87 MG cells was additionally followed up to 48 hours with exposure to different concentrations of CdCl₂ (1, 10 μM). Induction was a time dependent process throughout the period. Isoform III (identified by chromatographic separation of isoform III from I/II) was present at all exposure times, but only in traces with respect to the prevailing amounts of MT-I/II isoforms. So induction can be attributed to isoform I/II only.     

The intervention of antioxidant therapy on platelet adhesion and immunomodulation in experimental physical stress

During effort overstress the reactive oxygen species act chiefly on unsaturated lipids, inducing the formation of certain peroxidation products. We have investigated malondialdehide (MDA), platelet adhesion index, and immunological activation parameters during effort overstress and administration of vitamins E and C. Biochemical measurements were performed on erythrocytes and heart homogenate. In the vitamin E supplemented group, the platelet adhesion index was constantly correlated with the MDA level (p < 0.001). There is a protecting effect concerning the oxidative stress in animals pretreated with vitamin E and C, which is expressed through the diminution of the MDA quantity both in the erythrocyte and in the heart. The physical effort required by swimming led to a decrease in the NBT test values and in the activity of the serum complement. The steady administration of vitamin E in the effort overstress, due to its antioxidant properties, causes the progressive decrease in peroxidation and platelet adhesion.     

Physiological and Molecular Background of Maize Cold-Tolerance Enhancement with S-methylmethionine Salicylate

Low temperature is amongst the most influential abiotic stress factors, having deep impact on plant growth, yield and productivity. Studies on beneficial effects of certain biologically active substances, S-methylmethionine (SMM) and salicylic acid (SA) have provided a lot of valuable information regarding their role to counteract harmful effects of environmental stresses such as chilling. To obtain a more complex and stable defence compound with an extended range of stress-protective effect, the new derivative S-methylmethionine salicylate (MMS) was synthesised from the natural, biologically active substances SMM and SA. Since both original materials have complex stress-protective roles, the new compound was expected to combine the effects of original substances and to stabilise the unstable SMM in the new compound, thus providing an extended stress tolerance. Photosynthetic efficiency and accumulation of stress-related metabolites (polyamines and flavonoids) were measured in chilled and control plants, with and without MMS pretreatment, and expression changes of several genes involved in the cold stress response were analysed by quantitative real-time PCR (RT-qPCR) and a detailed microarray study. Our data show how the MMS combines the effect of SMM and SA on molecular level, causing numerous changes in the gene expression pattern and metabolite content. MMS gives rise to a better physiological condition, thus it could provide an alternative, environmental friendly way to enhance the plants defence mechanisms against stressors. As MMS is more stable than SMM, it promises easier, more long-lasting and more cost-effective usage in agriculture, with a complementing effect of SA.

     

A reversible decrease in ribulose 1,5‐bisphosphate carboxylase/oxygenase carboxylation activity caused by the aggregation of the enzyme's large subunit is triggered in response to the exposure of moderate irradiance‐grown plants to low irradiance

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) is highly regulated in response to fluctuations in the environment, including changes in irradiance. However, no complex data are available on Rubisco regulatory mechanisms triggered in plants which are submitted to moderate–low irradiance shift. Therefore, we investigated in a comprehensive way the changes at the level of amount of Rubisco protein, its structural organization and carboxylase activity of the holoenzyme as triggered by exposure of moderate irradiance‐grown Arabidopsis thaliana plants to low irradiance conditions. An exposure of moderate irradiance‐grown plants to low irradiance for a single photoperiod caused the exclusion of a certain pool of Rubisco under altered conditions owing to oxidative modifications resulting in the formation of protein aggregates involving Rubisco large subunit (LS). As a result, both initial and total Rubisco carboxylase activities were reduced, whereas Rubisco activation state remained largely unchanged. The results of the determination of reactive oxygen species indicated that a moderate/low irradiance transition had stimulated ¹O₂ accumulation and we strongly suggest that Rubisco oxidative modifications leading to formation of aggregates encompassing Rubisco‐LS were triggered by ¹O₂. When moderate irradiance regime was resumed, the majority of Rubisco‐LS containing aggregates tended to be resolubilized, and this allowed Rubisco carboxylation activities to be largely recovered, without changes in the activation state of the enzyme. In the longer term, these results allow us to better understand a complexity of Rubisco regulatory mechanisms activated in response to abiotic stresses and during recovery from the stresses.     

Regulation of the Rev1–pol ζ complex during bypass of a DNA interstrand cross‐link

DNA interstrand cross‐links (ICLs) are repaired in S phase by a complex, multistep mechanism involving translesion DNA polymerases. After replication forks collide with an ICL, the leading strand approaches to within one nucleotide of the ICL (“approach”), a nucleotide is inserted across from the unhooked lesion (“insertion”), and the leading strand is extended beyond the lesion (“extension”). How DNA polymerases bypass the ICL is incompletely understood. Here, we use repair of a site‐specific ICL in Xenopus egg extracts to study the mechanism of lesion bypass. Deep sequencing of ICL repair products showed that the approach and extension steps are largely error‐free. However, a short mutagenic tract is introduced in the vicinity of the lesion, with a maximum mutation frequency of ~1%. Our data further suggest that approach is performed by a replicative polymerase, while extension involves a complex of Rev1 and DNA polymerase ζ. Rev1–pol ζ recruitment requires the Fanconi anemia core complex but not FancI–FancD2. Our results begin to illuminate how lesion bypass is integrated with chromosomal DNA replication to limit ICL repair‐associated mutagenesis.     

Exploiting single-molecule transcript sequencing for eukaryotic gene prediction

We develop a method to predict and validate gene models using PacBio single-molecule, real-time (SMRT) cDNA reads. Ninety-eight percent of full-insert SMRT reads span complete open reading frames. Gene model validation using SMRT reads is developed as automated process. Optimized training and prediction settings and mRNA-seq noise reduction of assisting Illumina reads results in increased gene prediction sensitivity and precision. Additionally, we present an improved gene set for sugar beet (Beta vulgaris) and the first genome-wide gene set for spinach (Spinacia oleracea). The workflow and guidelines are a valuable resource to obtain comprehensive gene sets for newly sequenced genomes of non-model eukaryotes.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0729-7) contains supplementary material, which is available to authorized users.     

Integration of BpMADS4 on various linkage groups improves the utilization of the rapid cycle breeding system in apple

Rapid cycle breeding in apple is a new approach for the rapid introgression of agronomically relevant traits (e.g. disease resistances) from wild apple species into domestic apple cultivars (Malus × domestica Borkh.). This technique drastically shortens the long‐lasting juvenile phase of apple. The utilization of early‐flowering apple lines overexpressing the BpMADS4 gene of the European silver birch (Betula pendula Roth.) in hybridization resulted in one breeding cycle per year. Aiming for the selection of non‐transgenic null segregants at the end of the breeding process, the flower‐inducing transgene and the gene of interest (e.g. resistance gene) that will be introgressed by hybridization need to be located on different chromosomes. To improve the flexibility of the existing approach in apple, this study was focused on the development and characterization of eleven additional BpMADS4 overexpressing lines of four different apple cultivars. In nine lines, the flowering gene was mapped to different linkage groups. The differences in introgressed T‐DNA sequences and plant genome deletions post‐transformation highlighted the unique molecular character of each line. However, transgenic lines demonstrated no significant differences in flower organ development and pollen functionality compared with non‐transgenic plants. Hybridization studies using pollen from the fire blight‐resistant wild species accession Malus fusca MAL0045 and the apple scab‐resistant cultivar ‘Regia’ indicated that BpMADS4 introgression had no significant effect on the breeding value of each transgenic line.