Symbiotic Hydrogenase Activity in Bradyrhizobium sp. (Vigna) Increases Nitrogen Content in Vigna unguiculata Plants

Bradyrhizobium sp. (Lupinus) and Bradyrhizobium sp. (Vigna) mutants in which hydrogenase (hup) activity was affected were constructed and analyzed. Vigna unguiculata plants inoculated with the Bradyrhizobium sp. (Vigna) hup mutant showed reduced nitrogenase activity and also a significant decrease in nitrogen content, suggesting a relevant contribution of hydrogenase activity to plant yield.     

Drill-assisted genomic DNA extraction from <Emphasis Type="Italic">Botrytis cinerea</Emphasis>

Current DNA extraction protocols for genomic DNA from Botrytis cinerea almost always start with mycelium that has been reduced to powder with liquid N₂ in a mortar, and this makes their application to a large number of samples slow and cumbersome. Here we present an adaptation of an existing method [Möller et al. (1992) Nucleic Acids Res 20: 6115–6116] for which the initial steps have been modified, including the homogenization of the fungus with sand and the aid of a common household drill. This method allows the processing of large number of samples in much shorter times and generates an average of 4 μg DNA per sample, of sufficient quality for use in PCR and Southern blotting.     

Characterization of the antibody response against NeuGcGM3 ganglioside elicited in non-small cell lung cancer patients immunized with an anti-idiotype antibody

1E10 mAb is an anti-Id murine mAb (Ab2 mAb) specific for an Ab1 mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In preclinical studies, this Ab2 Ab was able to mimic NeuGc-containing gangliosides only in animals lacking expression of these Ags in normal tissues. In this study, we report on the immune responses elicited in 20 non-small cell lung cancer patients treated with 1 mg of aluminum hydroxide-precipitated 1E10 mAb. In the hyperimmune sera from 16 of 20 patients, a strong specific Ab response of both IgM and IgG isotypes against NeuGcGM3 ganglioside was observed. Patient immune sera were able to induce complement-independent cell death of NeuGcGM3-expressing X63 murine myeloma target cells. Significant immunoreactivity to NeuGcGM3 was still detected after the complete abrogation of the reactivity against 1E10 mAb by the adsorption of patient sera with this Ab. We hypothesize that Id(-)Ag(+) Abs could reflect the activation of an autologous idiotypic cascade into the patients. Both Id(+)Ag(+) and Id(-)Ag(+) fractions were separated by affinity chromatography and characterized. Although IgG isotype Abs were found in both fractions, IgM isotype Abs were found only in the Id(-)Ag(+) fraction. Both Id(+)Ag(+) and Id(-)Ag(+) Abs were able to specifically recognize and induce cell death in NeuGcGM3-expressing X63 myeloma target cells. Patients that developed IgG and/or IgM Abs against NeuGcGM3 showed longer median survival times.     

Human fucosyltransferase IX: specificity towards N-linked glycoproteins and relevance of the cytoplasmic domain in intra-Golgi localization

The alpha3-fucosyltransferase IX (FUT9) catalyses the transfer of fucose in an alpha3 linkage onto terminal type II (Galbeta4GlcNAc) acceptors, the final step in the biosynthesis of the Lewis(x) (Le(x)) epitope, in neurons. In this work, FUT9 cloned from NT2N neurons and overexpressed in HeLa cells (FUT9wt), was found to efficiently fucosylate asialoerythropoietin (asialoEPO), and bovine asialofetuin, but not sialylated EPO. Analysis by HPAEC-PAD and MALDI/TOF-MS revealed predominantly mono-fucosylation by FUT9wt of type II di-, tri- and tetraantennary N-glycans with proximal fucose, with and without N-acetylactosamine repeats from asialoEPO. Minor amounts of difucosylated structures were also found. The results suggested that FUT9 could fucosylate Le(x) carrier-glycoproteins in neurons. Furthermore, FUT9wt was found to be activated by Mn(2+) and it was capable of synthesizing Le(a), although to a lesser extent than Le(x) and Le(y). In vivo, HeLa cells transfected with FUT9wt expressed de novo Le(x), as detected by immunofluorescence microscopy. FUT9 was found to be a trans-Golgi and trans-Golgi network (TGN) glycosyltransferase from confocal immunofluorescence co-localization with the markers of the secretory pathway beta4-galactosyltransferase (trans-Golgi and TGN) and TGN-46 (TGN). Deletion of the cytoplasmic domain caused a shift to the cis-Golgi, thus suggesting that information for intra-Golgi localization is contained within the cytoplasmic domain.     

The Aspergillus nidulans phsB4 mutation alters colonial growth and development of the mould at acidic pH

The phsB4 mutant of the mould Aspergillus nidulans, identified as showing increased sensitivity to acid pH, is mitotically unstable and its conidia swell and lyse, forming protoplasts during germination and early development in shaken liquid cultures. On solid medium, we observed balloon-shaped hyphal swellings, a phenotype also exhibited by the chitin synthase gene (chsD) disruptants. We also observed that lysis was osmotically remediable with 0.5 M NaCl, but the balloon-shaped hyphal swelling was remedied in a pH-dependent way i.e., this phenotype was remedied only at pH values above 6.5. Based on the nature of our mutant selection, the pH sensitive phenotype of the selected strains, the known occurrence of hyphal swelling in cell wall mutants of A. nidulans, and the transformation with cosmids that hybridize to chsD gene, the phsB and chsD genes are possibly alleles.     

Enthalpic and entropic contributions mediate the role of disulfide bonds on the conformational stability of interleukin-4

The role of disulfide bridges in the structure, stability, and folding pathways of proteins has been the subject of wide interest in the fields of protein design and engineering. However, the relative importance of entropic and enthalpic contributions for the stabilization of proteins provided by disulfides is not always clear. Here, we perform a detailed analysis of the role of disulfides in the conformational stability of human Interleukin-4 (IL4), a four-helix bundle protein. In order to evaluate the contribution of two out of the three disulfides to the structure and stability of IL4, two IL4 mutants, C3T-IL4 and C24T-IL4, were used. NMR and ANS binding experiments were compatible with altered dynamics and an increase of the nonpolar solvent-accessible surface area of the folded state of the mutant proteins. Chemical and thermal unfolding experiments followed by fluorescence and circular dichroism revealed that both mutant proteins have lower conformational stability than the wild-type protein. Transition temperatures of unfolding decreased 14 degrees C for C3T-IL4 and 10 degrees C for C24T-IL4, when compared to WT-IL4, and the conformational stability, at 25 degrees C, decreased 4.9 kcal/mol for C3T-IL4 and 3.2 kcal/mol for C24T-IL4. Interestingly, both the enthalpy and the entropy of unfolding, at the transition temperature, decreased in the mutant proteins. Moreover, a smaller change in heat capacity of unfolding was also observed for the mutants. Thus, disulfide bridges in IL4 play a critical role in maintaining the thermodynamic stability and core packing of the helix bundle.     

The endo-beta-1,4-xylanase xyn11A is required for virulence in Botrytis cinerea

Phytopathogenic fungi can degrade xylan, an abundant hemicellulose in plant cell walls, by the coordinate action of a group of extracellular enzymes. Among these, endo-beta-1,4-xylanases carry out the initial breakdown by cleaving internal bonds in the polymer backbone. We have isolated and characterized a gene, xyn11A, coding for an endo-beta-1,4-xylanase belonging to family 11 of glycosyl hydrolases. xyn11A was shown to be induced by xylan and repressed by glucose and to be expressed in planta. The disruption of xyn11A caused only a moderate decrease, about 30%, in the level of extracellular endo-beta-1-4-xylanase activity and in the growth rate, with beechwood xylan as the only carbon source. However, deletion of the gene had a more pronounced effect on virulence, delaying the appearance of secondary lesions and reducing the average lesion size by more than 70%. Reintroducing the wild-type gene into the mutant strains reversed this phenotype back to wild type.     

The zebrafish (Danio rerio) caudal complex – a model to study vertebral body fusion

Impairment of segmentation during embryonic development leads to congenital fusion of vertebrae. Nevertheless, vertebral fusion can also occur during post‐embryonic life. Fusion can cause reduction in mobility and may be pathological, but it can also be part of normal development and mechanically required, such as in the teleost caudal skeleton, or in the tetrapod sacrum. Using a series of closely spaced ontogenetic stages of zebrafish, stained for mineralized (Alizarin red) and cartilaginous (Alcian blue) structures, we have characterized all fusions occurring during the formation of the caudal skeleton. The urostyle results from the vertebral fusion of the compound centrum preural1‐ural1 [PU1⁺+U1] and ural2 [U2⁺]. Based on developmental and morphological characters: (i) number of vestigial haemal arches, (ii) occasional presence of a haemal arch rudiment, (iii) occasional individuals with separate centra rudiments or distinct mineralization time points, and (iv) evidence for internal separation, we propose that the urostyle forms as a fusion product of five, and not three vertebral centra, as previously described. The last fusion to occur in development, between the compound centrum [PU1⁺+U1] and U2⁺, is a relatively slow process that typically occurs in Cypriniformes and Salmoniformes and is therefore considered reliable to monitor the fusion process. The vertebrae adjacent to the urostyle, preurals 2 and 3, are highly susceptible to fusion, and thus inadequate as a negative control to fusion, in contrast to trunk vertebrae, where fusion is never observed. With this we have established the basis for a new model to study vertebral fusion and to unravel cellular and molecular events underlying this process.     

Seasonal changes in daily metabolic patterns of tegu lizards (Tupinambis merianae) placed in the cold (17 degrees C) and dark

Abstract Oxygen consumption rate was measured continuously in young tegu lizards Tupinambis merianae exposed to 4 d at 25 degrees C followed by 7-10 d at 17 degrees C in constant dark at five different times of the year. Under these conditions, circadian rhythms in the rate of oxygen consumption persisted for anywhere from 1 d to the entire 2 wk in different individuals in all seasons except the winter. We also saw a progressive decline in standard oxygen consumption rate (at highly variable rates in different individuals) to a very low rate that was seasonally independent (ranging from 19.1 +/- 6.2 to 27.7 +/- 0.2 mL kg(-1) h(-1) across seasons). Although this degree of reduction appeared to take longer to invoke when starting from higher metabolic rates, tegu lizards reduced their metabolism to the low rates seen in winter dormancy at all times of the year when given sufficient time in the cold and dark. In the spring and summer, tegus reduced their standard metabolic rate (SMR) by 80%-90% over the experimental run, but only roughly 20%-30% of the total fall was due to the reduction in temperature; 70%-80% of the total fall occurred at constant temperature. By autumn, when the starting SMR on the first night at 25 degrees C was already reduced by 59%-81% (early and late autumn, respectively) from peak summer values, virtually all of the fall (63%-83%) in metabolism was due to the reduction in temperature. This suggests that the temperature-independent reduction of metabolism was already in place by autumn before the tegus had entered winter dormancy.