In-situ localization of chalcone synthase mRNA in pea root nodule development

In this paper studies on the role of flavonoids in pea root nodule development are reported. Flavonoid synthesis was followed by localizing chalcone synthase (CHS) mRNA in infected pea roots and in root nodules. In a nodule primordium, CHS mRNA is present in all cells of the primordium. Therefore it is hypothesized that the Rhizobium Nod factor induces cell division in the root cortex by stimulating the production of flavonoids that function as auxin transport inhibitors. In nodules CHS mRNA is predominantly present in a region at the apex of the nodule consisting of meristematic and cortical cells. These cells are not infected by Rhizobium. Therefore it is postulated that CHS plays a role in nodule development rather than in a defence response. In roots CHS mRNA is located at a similar position as in nodules, suggesting that CHS has the same function in both root and nodule development. When nodules are formed by mutants of Rhizobium leguminosarum bv. viciae that are unable to secrete β(1-2) glucan and to synthesize the O-antigen containing LPS I, CHS genes are also expressed in regions of the nodule that are infected by Rhizobium. It is postulated that the impaired development of nodules formed by these mutants is due to an induction of a plant defence response.     

Expression of the major tyrosyl phosphoprotein of 54 KDa in the integument of the medflyCeratitis capitata

Expression of a 54 kDa tyrosyl phosphorylated protein in epidermal cells during the third instar larval stage was followed. It was demonstrated that the 54 kDa protein moiety and its phosphorylated counterpart follow the same developmental profile. The system seems to be regulated only at the onset of the second moult, by an initial signal which regulates both the synthesis and phosphorylation of a 54 kDa protein. The continuous presence this protein in epidermal cells during the third instar stage, as well as during apolysis and histolysis, suggests that it might participate in cell activities taking place during this developmental period. However, the 54 kDa protein could no be involved in specific epidermal cell activities such as histolysis, melanization and sclerotization, since these activities occur only at specific times during the third instar stage.     

Structural variation in the antithrombin III binding site region and its occurrence in heparin from different sources

A tetrasaccharide possessing a biosynthetically permissible structural variability in and adjacent to the antithrombin III (ATIII) binding site has been isolated from heparin lyase depolymerized bovine lung heparin by using strong anion-exchange high-pressure liquid chromatography (SAX-HPLC). On the basis of two-dimensional 500-MHz 1H NMR experiments, including phase-sensitive correlated spectroscopy (COSY) and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY), and fast-atom bombardment mass spectrometry (FAB-MS), the primary structure of this tetrasaccharide was unambiguously established as delta UAp2S (1----4)-alpha-D-GlcNp2S6S(1----4)-beta-D-GlcAp(1----4)-alph a-D-GlcNp2S3S6S (where delta UA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid). The 1H NMR ROESY experiment proved to be particularly valuable in offering sequence information. Heparins from a variety of species and tissue sources were examined by oligosaccharide mapping using SAX-HPLC and gradient polyacrylamide gel electrophoresis. Two of these heparins are used as anticoagulants; they are porcine intestinal mucosal heparin and bovine lung heparin. The predominant ATIII-binding site in porcine heparin contained an N-acetylated glucosamine residue. We now report the structure of the predominant ATIII-binding site in bovine heparin as----4)-alpha-D-GlcNp2S6S(1----4)-beta-D-GlcAp(1----4)-alph a-D- GlcNp2S3S6S(1----4)-alpha-L-IdoAp2S(1----4)-alpha-D-GlcNp 2S6S(1----. This study shows the presence of one or both types of ATIII-binding-site variants in all of the heparins that were examined.     

A phylogenomic resolution for the taxonomy of Aegean green lizards

Lacerta pamphylica and Lacerta trilineata are two currently recognized green lizard species with a historically problematic taxonomy. In cases of tangled phylogenies, next-generation sequencing and double-digest restriction-site-associated DNA protocols can provide a wealth of genomic data and resolve difficult taxonomic issues. Here, we generated genome-wide SNPs and mitochondrial sequences, and applied molecular species delimitation approaches to provide a stable taxonomy for the Aegean green lizards. Mitochondrial gene trees, genetic cluster delimitation and population structure analyses converged into recognizing the populations of (a) L. pamphylica, (b) east Aegean islands, Anatolia and Thrace (diplochondrodes lineage), (c) central Aegean islands (citrovittata), and (d) remaining Balkan populations and islands (trilineata), as separate clusters. Phylogenomic analyses revealed a split into two major clades, east and west of the Aegean Barrier, unambiguously showing a sister–clade relationship between pamphylica and diplochondrodes, rendering L. trilineata paraphyletic. Species delimitation models were tested in a Bayesian framework using the genomic SNPs: lumping all populations into a single ‘species’ had the lowest likelihood but the current taxonomy was also outperformed by all other models. All lines of evidence support the Pamphylian green lizard as a valid species; thus, east Aegean L. trilineata should also be considered a distinct species under the name Lacerta diplochondrodes. Finally, evidence from the mitochondrial and nuclear genomes is overwhelmingly in favour of recognizing the morphologically distinct Cycladian green lizards as a distinct species. We propose their elevation to full species under the name Lacerta citrovittata. All remaining insular and continental populations of the Balkan Peninsula represent the species L. trilineata.     

Overproduction of d-xylose isomerase in Escherichia coli by cloning the d-xylose isomerase gene

The Escherichia coli d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) gene, xylA, has been cloned on various E. coli plasmids. However, it has been found that high levels of overproduction of the d-xylose isomerase, the protein product of the xylA gene, cannot be accomplished by cloning the intact gene on high copy-number plasmids alone. This is believed to be due to the fact that the expression of the gene through its natural promoter is highly regulated in E. coli. In order to overcome this, the xylA structural gene has been fused with other strong promoters such as tac and lac, resulting in the construction of a number of fused genes. Analysis of the E. coli transformants containing the fused genes, cloned on high copy-number plasmids, indicated that a 20-fold overproduction of the enzyme can now be obtained. It is expected that overproduction of the enzyme in E. coli can still be substantially improved through additional manipulation with recombinant DNA techniques.     

The in vivo preventive and therapeutic properties of curcumin in bile reflux‐related oncogenesis of the hypopharynx

Bile at strongly acidic pH exerts a carcinogenic effect on the hypopharynx, based upon recent pre‐clinical studies that support its role as an independent risk factor. We recently demonstrated in vitro that curcumin can prevent oncogenic profile of bile in human hypopharyngeal cells, by inhibiting NF‐κB. We hypothesize that topically applied curcumin to the hypopharynx can similarly block early oncogenic molecular events of bile, by inhibiting NF‐κB and consequently altering the expression of genes with oncogenic function. Using Mus musculus (C57Bl/6J), we topically applied curcumin (250 μmol/L; three times per day; 10 days) to the hypopharynx, 15 minutes before, 15 minutes after or in combination with bile acids (pH 3.0). Immunohistochemical analysis and qPCR revealed that topically applied curcumin either before, after or in combination with acidic bile exposure significantly suppressed its induced NF‐κB activation in regenerating epithelial cells, and overexpression of Rela, Bcl2, Egfr, Stat3, Wnt5a, Tnf, Il6, Ptgs2. Akt1 was particularly inhibited by curcumin when applied simultaneously with bile. We provide novel evidence into the preventive and therapeutic properties of topically applied curcumin in acidic bile‐induced early oncogenic molecular events in hypopharyngeal mucosa, by inhibiting NF‐κB, and shaping future translational development of effective targeted therapies using topical non‐pharmacologic inhibitors of NF‐κB.