Heteronuclear correlation experiments for the determination of one-bond coupling constants

Spin-state selective experiments, HSQC-/ and CT-HMQC-/, are proposed for the simple and rapid measurement of scalar one-bond coupling constants in two-dimensional,¹ H-detected ¹⁵N-¹H or¹³ C-¹H correlation experiments based on HSQC and HMQC schemes. Pairs of subspectra are obtained, containing either the high-field or the low-field component of the doublet representing the one-bond coupling constant. The subspectral editing procedure retains the full sensitivity of HSQC and HMQC spectra recorded without heteronuclear decoupling during data acquisition, with a spectral resolution similar to that of decoupled spectra.     

Phenology of Festuca pallescens in relation to topography in north-western Patagonia

Abstract. The phenological changes in populations of Festuca pallescens (St. Yves) Parodi at different topographic positions and exposure along an altitudinal gradient (600 - 1100 m) were investigated during two growing seasons in northwestern Patagonia. Stepwise multiple regression analysis was used to describe the relationship between phenology and environment during the entire growing season. Analysis of variance was also performed at each sample date to detect significant environmental factors influencing phenology at different sites. The sum of maximum air temperatures was identified as the environmental variable best correlated with the seasonal variation of phenological events of Festuca pallescens over the period of two growing seasons, explaining 93.2 % of the total variance. Significant differences between sites were observed at each sample date. Main effects of altitude and topographic position and two-way interactions between altitude and topographic position, and topographic position and exposure were also detected as significant. Phenology was delayed at increased altitude. Differences in phenology between topographic sites at the same altitude were not detected during the entire growing season and were only observed in the reproductive phase. At this time, the phenology was significantly delayed at high topographic positions on the slopes as compared with low and mid positions. At high altitudes in the valley (950 m a. s. 1.), where steep slopes and humid conditions prevail, phenology was delayed on western exposures and low positions. The results adequately summarize and quantify the effect of spatial and temporal environmental variation on the phenological development of Festuca pallescens in northwestern Patagonia.     

Protein-protein interactions between hepatitis C virus nonstructural proteins

Replication of the hepatitis C virus (HCV) genome has been proposed to take place close to the membrane of the endoplasmic reticulum in membrane-associated replicase complexes, as is the case with several other plus-strand RNA viruses, such as poliovirus and flaviviruses. The most obvious benefits of this property are the possibility of coupling functions residing in different polypeptidic chains and the sequestration of viral proteins and nucleic acids in a distinct cytoplasmic compartment with high local concentrations of viral components. Indeed, HCV nonstructural (NS) proteins were clearly colocalized in association with membranes derived from the endoplasmic reticulum. This observation, together with the demonstration of the existence of several physical interactions between HCV NS proteins, supports the idea of assembly of a highly ordered multisubunit protein complex(es) probably involved in the replication of the viral genome. The objective of this study, therefore, was to examine all potential interactions between HCV NS proteins which could result in the formation of a replication complex(es). We identified several interacting viral partners by using a glutathione S-transferase pull-down assay, by in vitro and ex vivo coimmunoprecipitation experiments in adenovirus-infected Huh-7 cells allowing the expression of HCV NS proteins, and, finally, by using the yeast two-hybrid system. In addition, by confocal laser scanning microscopy, NS proteins were clearly shown to colocalize when expressed together in Huh-7 cells. We have been able to demonstrate the existence of a complex network of interactions implicating all six NS proteins. Our observations confirm previously described associations and identify several novel homo- and heterodimerizations.     

Limited diversity of the immunoglobulin heavy chain variable domain of the emerald rockcod Trematomus bernacchii

To investigate the diversity of the immunoglobulin heavy chain variable domain of the cold adapted teleost Trematomus bernacchii, 45 cDNA clones, containing complete or partial sequences of rearranged VH/D/JH segments, were analysed. Clones were isolated from a spleen library constructed by 5' RACE or from an expression library previously constructed and immunoscreened with rabbit anti- T. bernacchii Ig heavy chain antibodies. VH sequences shared, on average, 79.9% nucleotide identity and defined only two gene families referred to as Trbe VH I and Trbe VH II, the latter comprising 89% of the VH sequences analysed in this study. A Southern blot analysis, performed with family specific probes, revealed that there are at least 25 genomic VH genes. A phylogenetic tree showed that Trbe VH I clustered with VH genes belonging to group D and Trbe VH II with those of group C. Four putative distinct D segments were found to contribute to the diversity of CDR3, which showed a high glycine content. The Shannon analysis revealed that FRs are very highly conserved. Of CDRs, CDR2 exhibits a mean entropy value higher than CDR1, contributing to variability in a significant manner. Moreover, eight distinct JH segments were identified. These findings provide several clues suggesting a limited diversity of the VH genes in the Antarctic teleost T. bernacchii.     

Metabolism of biodiesel-derived glycerol in probiotic Lactobacillus strains

Three probiotic Lactobacillus strains, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus delbrueckii, were tested for their ability to assimilate and metabolize glycerol. Biodiesel-derived glycerol was used as the main carbon and energy source in batch microaerobic growth. Here, we show that the tested strains were able to assimilate glycerol, consuming between 38 and 48 % in approximately 24 h. L. acidophilus and L. delbrueckii showed a similar growth, higher than L. plantarum. The highest biomass reached was 2.11 g?L^?1 for L. acidophilus, with a cell mass yield (Y _X/S) of 0.37 g?g^?1. L. delbrueckii and L. plantarum reached a biomass of 2.06 and 1.36 g?L^?1. All strains catabolize glycerol mainly through glycerol kinase (EC 2.7.1.30). For these lactobacillus species, kinetic parameters for glycerol kinase showed Michaelis–Menten constant (K _m) ranging from 1.2 to 3.8 mM. The specific activities for glycerol kinase in these strains were in the range of 0.18 to 0.58 U?mg?protein^?1, with L. acidophilus ATCC 4356 showing the maximum specific activity after 24 h of cultivation. Glycerol dehydrogenase activity was also detected in all strains studied but only for the reduction of glyceraldehyde with NADPH (K _m for DL-glyceraldehyde ranging from 12.8 to 32.3 mM). This enzyme shows a very low oxidative activity with glycerol and NADP⁺ and, most likely, under physiological conditions, the oxidative reaction does not occur, supporting the assumption that the main metabolic flux concerning glycerol metabolism is through the glycerol kinase pathway.     

DACH1 inhibits transforming growth factor-beta signaling through binding Smad4

The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, we hypothesized that DACH1 might play a similar cellular function. Herein, DACH1 was found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-beta (TGF-beta)-induced apoptosis. DACH1 repressed TGF-beta induction of AP-1 and Smad signaling in gene reporter assays and repressed endogenous TGF-beta-responsive genes by microarray analyses. DACH1 bound to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localized with NCoR in nuclear dotlike structures. NCoR enhanced DACH1 repression, and the repression of TGF-beta-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH was sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibited the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-beta signaling by interacting with NCoR and Smad4.     

Conidial anastomosis tubes in Colletotrichum

We describe the occurrence of special kinds of hyphae that create anastomoses directly between conidia. They can be found both in the laboratory and on infected plants. They first appear within asexual fruiting bodies approximately 15 days after conidiation has begun leading to the appearance of chains of connected conidia. Coincident with this we demonstrate in Colletotrichum lindemuthianum nuclear dynamics, including fragmentation, with cytoplasmic flow and passage of nuclei and organelles between conidia through the anastomosis tubes. We propose that conidial anastomosis tubes play an important role in the life cycle of these fungi.     

Coumarin inhibits the growth of carrot (Daucus carota L. cv. Saint Valery) cells in suspension culture

We used a carrot (Daucus carota L. cv. Saint Valery) cell suspension culture as a simplified model system to study the effects of the allelochemical compound coumarin (1,2 benzopyrone) on cell growth and utilisation of exogenous nitrate, ammonium and carbohydrates. Exposure to micromolar levels of coumarin caused severe inhibition of cell growth starting from the second day of culture onwards. At the same time, the presence of 50 mumol/L coumarin caused accumulation of free amino acids and of ammonium in the cultured cells, and stimulated their glutamine synthetase, glutamate dehydrogenase, glucose-6-phosphate dehydrogenase and phosphoenolpyruvate carboxylase activities. Malate dehydrogenase, on the other hand, was inhibited under the same conditions. These effects were interpreted in terms of the stimulation of protein catabolism and/or interference with protein biosynthesis induced by coumarin. This could have led to a series of compensatory changes in the activities of enzymes linking nitrogen and carbon metabolism. Because coumarin seemed to abolish the exponential phase and to accelerate the onset of the stationary phase of cell growth, we hypothesise that such allelochemical compounds may act in nature as an inhibitor of the cell cycle and/or as a senescence-promoting substance.