N-Benzoyl anthranilic acid derivatives as selective inhibitors of aldo-keto reductase AKR1C3

Human aldo-keto reductases AKR1C1-AKR1C3 are involved in the biosynthesis and inactivation of steroid hormones and prostaglandins and thus represent attractive targets for the development of new drugs. We synthesized a series of N-benzoyl anthranilic acid derivatives and tested their inhibitory activity on AKR1C enzymes. Our data show that these derivatives inhibit AKR1C1-AKR1C3 isoforms with low micromolar potency. In addition, five selective inhibitors of AKR1C3 were identified. The most promising inhibitors were compounds 10 and 13, with IC(50) values of 0.31μM and 0.35μM for AKR1C3, respectively.     

The comparison of seed protein patterns within the genusArachis by polyacrylamide gel electrophoresis

The seed protein patterns of 12Arachis species were compared by polyacrylamide gel electrophoresis (PAGE), similarities between patterns were measured by the Jaccard index. Results obtained confirm the close relationships established between members of the genus on morphological grounds and support the more recent classification schemes.A. villosa andA. correntina could well be regarded as distinct species on grounds of protein differences whileA. macedoi andA. villosulicarpa (although members of the same section, Extranervosae) show considerable differentiation of their protein patterns. Surprisingly, the formA. ×batizogaea showed less similarity in protein pattern to those of its parental species than might have been expected. The principle value of seed protein pattern data appears to be in distinguishing species within sections.     

Further cytogenetic analyses of the Croatian triploid shallot “Ljutika” (Allium cepa var.viviparum, Alliaceae) and its comparison with the Indian triploid “Pran”

Feulgen and silver-stained karyotypes and meiosis of two triploid viviparous onion forms (Allium cepa var.viviparum), the Croatian Ljutika and the Indian Pran, were comparatively analyzed. The results of chromosome measurements show that Ljutika and Pran are karyologically not identical, although significant similarities were found in the morphology of their chromosomes. Five geographically distant clones of Ljutika showed good agreement in the number and gross morphology of the chromosomes and in the number and position of NORs and interphase nucleoli. Heterotrivalents were predominant in meiosis of Ljutika but a relatively high frequency of higher multivalents together with univalents and bivalents were also observed. The relationship between Ljutika and Pran and their possible origin are discussed.     

Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells

The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system.     

Targeting the insulin receptor with hormone and peptide dimers

Insulin is a key hormone involved in the regulation of overall energetic homeostasis of the organism. The dimeric character of the receptor for insulin evokes ideas about its activation or inhibition with peptide dimers that could either trigger or block the structural transition of the insulin receptor, leading to its activation. Herewith, we present the chemical engineering and biological characterization of several series of insulin dimers or dimers of specific peptides that should be able to bind receptors for insulin or insulin growth factor 1. The hormones or peptides in the dimers were interconnected with different linkers, consisting of triazole moieties and 3, 6, 8, 11, or 23 polyethylene glycol units. The prepared dimers were weaker in binding to insulin receptors than human insulin. However, some of the insulin dimers showed preferential binding specificity toward the isoform A of the insulin receptor, and the insulin dimers also stimulated the insulin receptor more strongly than would be consistent with their binding affinities. Our results suggest that designing insulin dimers may be a promising strategy for modulating the ability of the hormone to activate the receptor or to alter its specificity toward insulin receptor isoforms.     

The Rep20 Replication Initiator From the pAG20 Plasmid of Acetobacter aceti

In the previously isolated pAG20 plasmid from the Acetobacter aceti CCM3610 strain, the Rep20 protein was characterized as a main replication initiator. The pAG20 plasmid origin was localized in the vicinity of the rep20 gene and contained two 21-nucleotide-long iteron sequences, two 13-nucleotide-long direct repeats, and a DnaA-binding site. Electrophoretic mobility shift assay and nonradioactive fragment analysis confirmed that the Rep20 protein interacted with two direct repeats (5′-TCCAAATTTGGAT′-3′) and their requirement during plasmid replication was verified by mutagenesis. Although the association could not be validated of the DnaA protein of from the host cells of Escherichia coli with the plasmid-encoded replication initiator that usually occurs during replication initiation, Rep20 was able to form dimeric structures by which it could bind the sequence of the rep20 gene and autoregulate its own expression. Targeted mutagenesis of the Rep20 protein revealed the importance of the third α-helix and ⁶³Lys, specifically during DNA binding. The second, closely adjacent β-sheet also took part in this process in which ⁵²Asn played a significant role.     

Exogenously applied 20-hydroxyecdysone increases the net photosynthetic rate but does not affect the photosynthetic electron transport or the content of photosynthetic pigments in Tetragonia tetragonioides L.

Phytoecdysteroids are steroid compounds present in many plant species (sometimes in rather large amounts), but their biological role is still far from being clear. We have found that the exogenous application of 20-hydroxyecdysone (20E) to leaves of Tetragonia tetragonioides L. causes stimulation of its net photosynthetic rate (P _N) but does not positively affect the photosynthetic electron transport or the content of photosynthetic pigments. The increase in P _N was observed shortly after 20E treatment and was statistically significant during the 4th and 6th hours after treatment but not later, which could be perhaps caused by a strictly short-term window of opportunity for ecdysteroids to significantly affect photosynthetic processes. To our knowledge, these results are the first to suggest a new potential biological function of phytoecdysteroids—regulation of photosynthesis.     

The roles of carbonic anhydrases IX and XII in cancer cell adhesion,migration, invasion and metastasis

The main function of carbonic anhydrases (CAs) in cancer cells is the pH regulation through a conversion of H₂O and CO₂ to H⁺ and HCO₃⁻. However, the data of in vitro and in vivo studies have demonstrated that transmembrane isoforms of CA IX and CA XII are involved in various steps of cancer cell migration, invasion and metastasis. According to literature, inhibition of these CAs can affect the expression of multiple proteins. Some scientific groups have reported the possible interactions between CA IX and E-cadherin–catenin system, CA IX and integrins, CA IX, CA XII and ion transporters, which all are highly involved in cell-to-cell adhesion, the formation of membrane protrusions and focal adhesions. Nevertheless, CA IX and CA XII have a high impact on tumour growth and metastases formation. The data discussed in this review are quite recent. It highly support the role of CA IX and CA XII in various cancer metastasis processes through their interactions to other invasion proteins. Nevertheless, all findings show the great potential of these CAs in the context of research and application in clinical use.     

Euhemerobe Kalk-Kieferngesellschaften der Kleinen Karpaten

Man made stands withPinus nigra Arn. were studied on limestone bedrock. They were planted out on biotops of previous calciphilous beech or xerothermophilous oak forests. The analysis of present phytocenological composition and the changes in ecological conditions, especially of nitrogen content in the soil, conduce to their classification, following the conception ofHada? andSofron (1980), as a new euhemerobic forest community, associationClematidi vitalbae-culti-Pinetum nigrae.