Synthesis and conformational analysis of methyl 3-amino-2,3-dideoxyhexopyranosiduronic acids, new sugar amino acids, and their diglycotides

The synthesis of methyl 3-azido- and 3-amino-2,3-dideoxyhexopyranosiduronic acids and their methyl esters with the -alpha,beta-D-arabino-, -alpha,beta-D-ribo, and -alpha,beta-L-lyxo configurations is presented. The conformations of the synthesized sugar amino acids and their precursors are discussed on the basis of 1H NMR data. The influence of the 5-carboxyl group on the pyranose ring conformation is assessed, and the bonding of the monosugar amino acids into dimeric glycotides, using conventional solution-phase peptide syntheses, is reported.     

New data on the cytology of parthenogenetic weevils (Coleoptera,Curculionidae)

Parthenogenesis and, in particular, polyploidy are rare in animals. A number of cases, known among weevils, represent apomictic parthenogenesis-a reproductive mode in which eggs undergo one maturation division, the chromosomes divide equationally, and no reduction takes place. Among parthenogenetic weevils there are two diploids, 48 triploids, 18 tetraploids, six pentaploids, three hexaploids and one decaploid. Eight examined parthenogenetic species are triploids with 33 chromosomes of different morphology, confirming that triploidy is the most common level of ploidy in weevils. The karyotypes are heterogeneous with the presence of meta-, submeta-, subtelo- and acrocentric chromosomes. The C-banding method showed that only two species possess a large amount of heterochromatin visible as a band around the centromere during mitotic metaphase. This agrees with observations that weevils are characterized by a small amount of heterochromatin, undetectable in metaphase plates after C-banding. In three species an atypical course of apomictic oogenesis occurs with stages similar to meiosis, in which chromosomes form bivalents and multivalent clusters. This association of chromosomes probably represents the remnants of meiosis, although these events have nothing to do with recombination. The results support the hypothesis that the evolution of apomictic parthenogenesis in weevils has proceeded through a stage of automixis.     

Interactions between stressful environment and gene deletions alleviate the expected average loss of fitness in yeast

The conjecture that the deleterious effects of mutations are amplified by stress or interaction with one another remains unsatisfactorily tested. It is now possible to reapproach this problem systematically by using genomic collections of mutants and applying stress-inducing conditions with a well-recognized impact on metabolism. We measured the maximum growth rate of single- and double-gene deletion strains of yeast in several stress-inducing treatments, including poor nutrients, elevated temperature, high salinity, and the addition of caffeine. The negative impact of deletions on the maximum growth rate was relatively smaller in stressful than in favorable conditions. In both benign and harsh environments, double-deletion strains grew on average slightly faster than expected from a multiplicative model of interaction between single growth effects, indicating positive epistasis for the rate of growth. This translates to even higher positive epistasis for fitness defined as the number of progeny. We conclude that the negative impact of metabolic disturbances, regardless of whether they are of environmental or genetic origin, is absolutely and relatively highest when growth is fastest. The effect of further damages tends to be weaker. This results in an average alleviating effect of interactions between stressful environment and gene deletions and among gene deletions.     

Localization of organic cation/carnitine transporter (OCTN2) in cells forming the blood-brain barrier

Carnitine β-hydroxy-γ-(trimethylammonio)butyrate – a compound necessary in the peripheral tissues for a transfer of fatty acids for their oxidation within the cell, accumulates in the brain despite low β-oxidation in this organ. In order to enter the brain, carnitine has to cross the blood–brain barrier formed by capillary endothelial cells which are in close interaction with astrocytes. Previous studies, demonstrating expression of mRNA coding two carnitine transporters – organic cation/carnitine transporter 2 (OCTN2) and B^0,+ in endothelial cells, did not give any information on carnitine transporters polarity in endothelium. Therefore more detailed experiments were performed on expression and localization of a high affinity carnitine transporter OCTN2 in an in vitro model of the blood–brain barrier by real-time PCR, western blot analysis, and immunocytochemistry. The amount of mRNA was comparable in endothelial cells and kidney, when referred to house-keeping genes, it was, however, significantly lower in astrocytes. Polarity of OCTN2 localization was further studied in an in vitro model of the blood–brain barrier with use of anti-OCTN2 antibodies. Z -axis analysis of the confocal microscope pictures of endothelial cells, with anti-P-glycoprotein antibodies as the marker of apical membrane, showed OCTN2 localization at the basolateral membrane and in the cytoplasmic region in the vicinity of nuclei. Localization of OCTN2 suggest that carnitine can be also transported from the brain, playing an important role in removal of certain acyl esters.     

Uniparental loss of ribosomal DNA in the allotetraploid grass Zingeria trichopoda (2n = 8).

Analysis of the grass Zingeria trichopoda (2n = 8, 2C = 5.3 pg) revealed a dynamic evolution with the following characteristics. (i) Genomic in situ hybridization (GISH) demonstrates that Z. trichopoda evolved from an interspecific hybrid involving a species like contemporary Zingeria biebersteiniana (2n = 4) and a second species with a similar low number of chromosomes. The nucleus of Z. trichopoda is spatially organized at the genome level and the two parental genomes occupy distinct and separate domains of lateral arrangements. (ii) The copy number of the Z. biebersteiniana specific pericentromeric tandem repeat family Zbcen1 is drastically reduced in Z. trichopoda. (iii) GISH in combination with labeled rDNA sequences simultaneously discriminated the two parental genomes and the corresponding 5S and 45S rDNA sites. Hence, following allopolyploidization of Z. trichopoda the Z. biebersteiniana like parental chromosomes probably underwent drastic loss of 45S rDNA. This could have arisen either through the loss of Z. biebersteiniana derived 45S rDNA or through Z. trichopoda genome-wide homogenization of Z. biebersteiniana type 45S rDNA and subsequent elimination of 45S rDNA loci from Z. biebersteiniana derived chromosomes. Finally, 5S rDNA loci are present in both subgenomes of Z. trichopoda and the chromosomal position of these loci is similar for both Z. biebersteiniana and the Z. biebersteiniana like parental genome of Z. trichopoda.     

Antifungal activity of alkyl and heterocyclic aza-derivatives of gossypol as well as their complexes with NaClO4 against Fusarium oxysporum f. sp. lupini

Eight alkyl and six heterocyclic aza-derivatives of gossypol (2–15) have been synthesized using gossypol (1) extracted from Gossypium Herbaceum cottonseeds. The ability of gossypol aza-derivatives to form complexes with NaClO₄ has been investigated by electrospray ionisation (ESI) mass spectra recorded in the positive and negative ion detection modes. The gossypol aza-derivatives have been characterized by FT-IR, ¹H and ¹³C NMR spectroscopic methods and subsequently tested for their antifungal properties against Fusarium oxysporum. Four alkyl aza-derivatives (2–5), present in the enamine–enamine tautomeric form, have shown activity comparable or higher than that of gossypol against this fungus. To improve the antifungal activity the complexes of the most active compounds 2–5 with NaClO₄ were prepared. Complexes of 2 and 5 with NaClO₄ have shown antifungal activity higher than that of the uncomplexed compounds.     

Rosetta Broker for membrane protein structure prediction: concentrative nucleoside transporter 3 and corticotropin-releasing factor receptor 1 test cases

Background

Membrane proteins are difficult targets for structure prediction due to the limited structural data deposited in Protein Data Bank. Most computational methods for membrane protein structure prediction are based on the comparative modeling. There are only few de novo methods targeting that distinct protein family. In this work an example of such de novo method was used to structurally and functionally characterize two representatives of distinct membrane proteins families of solute carrier transporters and G protein-coupled receptors. The well-known Rosetta program and one of its protocols named Broker was used in two test cases. The first case was de novo structure prediction of three N-terminal transmembrane helices of the human concentrative nucleoside transporter 3 (hCNT3) homotrimer belonging to the solute carrier 28 family of transporters (SLC28). The second case concerned the large scale refinement of transmembrane helices of a homology model of the corticotropin-releasing factor receptor 1 (CRFR1) belonging to the G protein-coupled receptors family.

Results

The inward-facing model of the hCNT3 homotrimer was used to propose the functional impact of its single nucleotide polymorphisms. Additionally, the 100 ns molecular dynamics simulation of the unliganded hCNT3 model confirmed its validity and revealed mobility of the selected binding site and homotrimer interface residues. The large scale refinement of transmembrane helices of the CRFR1 homology model resulted in the significant improvement of its accuracy with respect to the crystal structure of CRFR1, especially in the binding site area. Consequently, the antagonist CP-376395 could be docked with Autodock VINA to the CRFR1 model without any steric clashes.

Conclusions

The presented work demonstrated that Rosetta Broker can be a versatile tool for solving various issues referring to protein biology. Two distinct examples of de novo membrane protein structure prediction presented here provided important insights into three major areas of protein biology. Namely, the dynamics of the inward-facing hCNT3 homotrimer system, the structural changes of the CRFR1 receptor upon the antagonist binding and finally, the role of single nucleotide polymorphisms in both, hCNT3 and CRFR1 proteins, were investigated.