Chromosomal and isozyme studies of Nicotiana tabacum — Glycine max hybrid cell lines

Summary The chromosomal stability of a number of somatic hybrids derived from soybean (Glycine max (L.) Merr.) and Nicotiana tabacum var. Xanthi were investigated. Several of the hybrid cell lines retained more than half the complement of N. tabacum chromosomes after 7 months of culturing. A number of chromosomal abnormalities were observed. The hybrids were positively identified by employing isozyme analysis of several dehydrogenases and aspartate aminotransferase.NRCC No. 20130     

The development and characterization of an E. coli O25B bioconjugate vaccine

Extraintestinal pathogenic Escherichia coli (ExPEC) cause a wide range of clinical diseases such as bacteremia and urinary tract infections. The increase of multidrug resistant ExPEC strains is becoming a major concern for the treatment of these infections and E. coli has been identified as a critical priority pathogen by the WHO. Therefore, the development of vaccines has become increasingly important, with the surface lipopolysaccharide constituting a promising vaccine target. This study presents genetic and structural analysis of clinical urine isolates from Switzerland belonging to the serotype O25. Approximately 75% of these isolates were shown to correspond to the substructure O25B only recently described in an emerging clone of E. coli sequence type 131. To address the high occurrence of O25B in clinical isolates, an O25B glycoconjugate vaccine was prepared using an E. coli glycosylation system. The O antigen cluster was integrated into the genome of E. coli W3110, thereby generating an E. coli strain able to synthesize the O25B polysaccharide on a carrier lipid. The polysaccharide was enzymatically conjugated to specific asparagine side chains of the carrier protein exotoxin A (EPA) of Pseudomonas aeruginosa by the PglB oligosaccharyltransferase from Campylobacter jejuni. Detailed characterization of the O25B-EPA conjugate by use of physicochemical methods including NMR and GC-MS confirmed the O25B polysaccharide structure in the conjugate, opening up the possibility to develop a multivalent E. coli conjugate vaccine containing O25B-EPA.

     

The role of transbronchial lung biopsy in the diagnosis of solitary pulmonary nodule

Transbronchial lung biopsy (TBLB) is a well-recognized diagnostic technique in diffuse interstitial lung diseases, but it is not considered to be the first choice in investigation of solitary pulmonary nodules (SPN). The main idea of this study was to increase the sensitivity of bronchoscopy using multiple techniques, especially TBLB, thus to avoid more aggressive diagnostic procedures. The objective of this prospective study was to evaluate the efficacy and safety of TBLB in the diagnosis of SPN, in comparison with other bronchoscopic techniques. Fifty patients with chest x-ray finding consistent with SPN underwent bronchoscopy with bronchial washing, brushing, bronchoalveolar lavage (BAL) and TBLB were included in this study. Thirty-one patients suffered from malignant tumors, while 19 patients had nonmalignant lesions. TBLB achieved overall diagnostic sensitivity of 62%, BAL of 29%, bronchial brushing of 16% and washing of 6%. Combining all techniques together, bronchoscopy had overall sensitivity of 86%. Concerning malignant lesions, TBLB had a sensitivity of 65%, specificity of 100%, and accuracy of 82%. TBLB had a significantly better yield for lesions with a diameter > or = 25 mm than for lesions of < 25 mm (sensitivity of 82% and 53% respectively, p < 0.05). Diagnostic yield improved significantly with the increasing number of specimens (less than 3 specimens: sensitivity 59%, 3 or more specimens: sensitivity 87%, p < 0.05). Complications of TBLB occurred in 2 (4%) patients: 1 incomplete pneumothorax and 1 hemorrhage. According to the results, we conclude that TBLB is an accurate and safe technique for the diagnosis of pulmonary solitary nodule with a diameter equal or greater than 25 mm.     

Rapid generation of a representative ensemble of N-glycan conformations

Glycosylated proteins are ubiquitous components of extracellular matrices and cellular surfaces where their oligosaccharide moieties are implicated in a wide range of cell-cell and cell-matrix recognition events. Glycans constitute highly flexible molecules. Only a small number of glycan X-ray structures is available for which sufficient electron density for an entire oligosaccharide chain has been observed. An unambiguous structure determination based on NMR-derived geometric constraints alone is often not possible. Time consuming computational approaches such as Monte Carlo calculations and molecular dynamics simulations have been widely used to explore the conformational space accessible to complex carbohydrates. The generation of a comprehensive data base for N-glycan fragments based on long time molecular dynamics simulations is presented. The fragments are chosen in such a way that the effects of branched N-glycan structures are taken into account. The prediction database constitutes the basis of a procedure to generate a complete set of all possible conformations for a given N-glycan. The constructed conformations are ranked according to their energy content. The resulting conformations are in reasonable agreement with experimental data. A web interface has been established (http://www.dkfz.de/spec/glydict/), which enables to input any N-glycan of interest and to receive an ensemble of generated conformations within a few minutes.     

Lower calorie intake enhances muscle insulin action and reduces hexosamine levels

Previous studies have demonstrated enhanced insulin sensitivity in calorie-restricted [CR, fed 60% ad libitum (AL) one time daily] compared with AL-fed rats. To evaluate the effects of reduced food intake, independent of temporal differences in consumption, we studied AL (unlimited food access)-fed and CR (fed one time daily) rats along with groups temporally matched for feeding [fed 3 meals (M) daily]: MAL and MCR, eating 100 and 60% of AL intake, respectively. Insulin-stimulated glucose transport by isolated muscle was increased in MCR and CR vs. AL and MAL; there was no significant difference for MCR vs. CR or MAL vs. AL. Intramuscular triglyceride concentration, which is inversely related to insulin sensitivity in some conditions, did not differ among groups. Muscle concentration of UDP-N-acetylhexosamines [end products of the hexosamine biosynthetic pathway (HBP)] was lower in MCR vs. MAL despite unaltered glutamine-fructose-6-phosphate aminotransferase activity (rate-limiting enzyme for HBP). These results indicate that the CR-induced increase in insulin-stimulated glucose transport in muscle is attributable to an altered amount, not timing, of food intake and is independent of lower triglyceride concentration. They further suggest that enhanced insulin action might involve changes in HBP.     

The interleukin-1 receptor antagonist gene and the inhibitor of kappa B-like protein gene polymorphisms are not associated with myocardial infarction in Slovene population with type 2 diabetes

In this study we investigated the association of the interleukin-1 receptor antagonist gene variable number tandem repeat (IL1RN VNTR) polymorphism and of the inhibitor of kappa B-like protein (IKBL) gene polymorphism with myocardial infarction (MI) in a group of patients with type 2 diabetes. The IL1RN VNTR and the IKBL+ 738T > C gene polymorphisms were tested in 374 Caucasians: 151 cases with MI and 223 subjects with no history of coronary artery disease. The IL1RN VNTR polymorphism was not a risk factor for MI in Caucasians with type 2 diabetes (genotype 22 vs. the rest: odds ratio (OR) 1.6; 95% confidence interval (CI) = 0.8-3.5; p = 0.2). We also failed to demonstrate that IKBL+ 738T > C gene polymorphism was associated with MI in patients with type 2 diabetes (OR = 0.9; 95% CI = 0.3-2.6; p = 0.9). We provide evidence that the IL1RN VNTR and the IKBL + 738T > C gene polymorphisms are not risk factors for MI in Caucasians with type 2 diabetes.     

Thrombin inhibitors with lipid peroxidation and lipoxygenase inhibitory activities

Vascular oxidative stress, endothelial injury, and thrombosis are intertwined processes that display a synergistic pathological effect in many cardiovascular diseases. Antithrombotic therapy with anticoagulant and/or antiplatelet agents, combined with interventions against vascular oxidative stress and/or inflammation, both boosting endothelial antithrombotic potential, could display a synergistic action in the treatment of thrombosis. Of the compounds 10a-h and 11a-d, shown to possess thrombin inhibitory activity, 11a-d were found to display radical scavenging activity, 10a, 10d, and 10f were demonstrated to inhibit lipid peroxidation of linoleic acid, and 10b and 10h inhibited soybean lipoxygenase. The observed combination of thrombin inhibition with lipid peroxidation and/or lipoxygenase inhibitory activity makes compounds 10 and 11 interesting candidates for further investigations towards multiple antithrombotic drugs.