Expression of wild-type GBSS transgenes in the off-spring of partially and fully complementedamylose-free transformants of potato

Theamylose-free (amf) potato mutant can easily be complemented through introduction of the wild-type gene coding for granule-bound starch synthase (GBSS). After iodine staining the starch of theamf mutant is red whereas that of the wild type and the complementedamf mutant is blue. The level of complementation of selected transformants and their sexual off-spring after backcrossing withamf was investigated using sporophytic tuber cells and gametophytic microspore cells. Two diploid and two tetraploid transformants with full complementation demonstrated the expected segregation patterns of 1:1 (one active insert) or 3:1 (two independently segregating active inserts) in the microspores and in the F1 offspring based on staining of tubers. All expected genotypes in the F1 generation were found, based on microspore segregation patterns of the individual F1 plants. Two transformants with partial complementation (mixed phenotypes) were investigated. One of them, B1, was tetraploid and duplex for the GBSS insert, which had originated through mitotic doubling of the transformed diploid cells. In the F1 generation three phenotypic classes were found:amf, fully complemented and partially complemented. The latter two classes exist independently of a simplex or duplex gene status. The second transformant with partial complementation, B10, appeared to have a complex molecular composition. One cluster of five transgenes caused the partial complementation. Fully and partially complemented phenotypic classes were found after crossing B10 with theamf mutant. Indications were found that the ploidy level of the tissue in which the genes were introduced and expressed played an important role. Firstly, partial complementation was found after transformation of the diploid and not of the tetraploidamf genotypes. Secondly, the level of complementation was higher in tissue with lower ploidy levels, as illustrated by the colour of the starch inin vitro tubers (2x–4x cells) versus field-grown tubers (16x–64x).     

Anaerobic degradation of volatile fatty acids at different sulphate concentrations

The effect of sulfate on the anaerobic breakdown of mixtures of acetate, propionate and butyrate at three different sulfate to fatty acid ratios was studied in upflow anaerobic sludge blanket reactors. Sludge characteristics were followed with time by means of sludge activity tests and by enumeration of the different physiological bacterial groups. At each sulfate concentration acetate was completely converted into methane and CO₂, and acetotrophic sulfate-reducing bacteria were not detected. Hydrogenotrophic methanogenic bacteria and hydrogenotrophic sulfate-reducing bacteria were present in high numbers in the sludge of all reactors. However, a complete conversion of H₂ by sulfate reducers was found in the reactor operated with excess sulfate. At higher sulfate concentrations, oxidation of propionate by sulfate-reducing bacteria became more important. Only under sulfate-limiting conditions did syntrophic propionate oxidizers out-compete propionate-degrading sulfate reducers. Remarkably, syntrophic butyrate oxidizers were well able to compete with sulfate reducers for the available butyrate, even with an excess of sulfate. Correspondence to: A. Visser     

Intracerebral Hemorrhages in Adults with Community Associated Bacterial Meningitis in Adults: Should We Reconsider Anticoagulant Therapy?

Objective

To study the incidence, clinical presentation and outcome of intracranial hemorrhagic complications in adult patients with community associated bacterial meningitis.

Methods

Nationwide prospective cohort study from all hospitals in the Netherlands, from 1 March 2006, through 31 December 2010.

Results

Of the 860 episodes of bacterial meningitis that were included, 24 were diagnosed with intracranial hemorrhagic complications: 8 upon presentation and 16 during clinical course. Clinical presentation between patients with or without intracranial hemorrhage was similar. Causative bacteria were Streptococcus pneumoniae in 16 patients (67%), Staphylococcus aureus in 5 (21%), Pseudomonas aeruginosa and Listeria monocytogenes both in 1 patient (4%). Occurrence of intracranial hemorrhage was associated with death (63% vs. 15%, P<0.001) and unfavorable outcome (94% vs. 34%, P<0.001). The use of anticoagulants on admission was associated with a higher incidence of intracranial hemorrhages (odds ratio 5.84, 95% confidence interval 2.17–15.76).

Conclusion

Intracranial hemorrhage is a rare but devastating complication in patients with community-associated bacterial meningitis. Since anticoagulant therapy use is associated with increased risk for intracranial hemorrhage, physicians may consider reversing or temporarily discontinuing anticoagulation in patients with bacterial meningitis.     

Influence of ferulic acid on the production of feruloyl esterases by Aspergillus niger

Extracellular feruloyl esterases from the filamentous fungus Aspergillus niger are induced by growth on oat spelt xylan (OSX), which contains no detectable esterified ferulic acid. FAE-III accounted for most of the feruloyl esterase activity. Addition of free ferulic acid to OSX at the start of the culture induced FAE-III secretion a further 2.3-fold, and also induced other feruloyl esterases which could not be ascribed to FAE-III. Wheat bran- (WB)-grown cultures, containing 1% (m/v) ester-linked ferulic acid, gave almost identical FAE-III and total feruloyl esterase activities as the cultures grown on OSX plus ferulic acid. De-esterification of WB yielded less total feruloyl esterase, and 2.4-fold less FAE-III, compared to untreated WB. A slightly modified form of FAE-III was produced on de-esterified WB. These results show that production of FAE-III does not absolutely require ferulic acid. However, production is stimulated by the presence of free ferulic acid through increased expression, and is reduced by the removal of esterified ferulic acid from the growth substrate.     

Separation of spleen colony-forming units and prothymocytes by use of a monoclonal antibody detecting an H-2K determinant

The density of H-2K antigens was determined on both the mouse hemopoietic stem cell, using an assay for spleen colony-forming units (CFU-S), and the prothymocyte, using a thymus repopulation assay. This was done by light-activated cell sorting of bone marrow cells labeled first with a biotinylated antibody against H-2Kk and then with avidin-fluorescein isothiocyanate. Almost all CFU-S were found to be present among the 4% bone marrow cells with high forward light scatter (FLS), low perpendicular light scatter (PLS), and bright immunofluorescence. Thymus regeneration by this brightly fluorescent fraction was delayed 3 days compared to thymus regeneration by unsorted cells, although the same number of CFU-S was present in each cell suspension. This delay indicates that differentiation from CFU-S to prothymocytes takes 3 days. The fraction of cells in the FLS/PLS window with dull anti-H-2Kk fluorescence contained few CFU-S and gave rise to a transient thymus regeneration. These findings indicate that the prothymocyte carries fewer H-2K antigens than does the CFU-S. The H-2K antigen is a marker with which CFU-S and prothymocytes can be separated. Therefore, during early T-cell differentiation, the number of H-2K molecules on the cell surface decreases (CFU-S----prothymocyte----cortical thymocyte). During maturation of T cells, a reexpression of H-2K molecules occurs, since lymph node cells and spleen cells were shown to be brightly positive for H-2K antigen.     

Functional and structural characterization of a synthetic peptide representing the N-terminal domain of prokaryotic pyruvate dehydrogenase

A synthetic peptide (Nterm-E1p) is used to characterize the structure and function of the N-terminal region (amino acid residues 4-45) of the pyruvate dehydrogenase component (E1p) from the pyruvate dehydrogenase multienzyme complex (PDHC) from Azotobacter vinelandii. Activity and binding studies established that Nterm-E1p specifically competes with E1p for binding to the dihydrolipoyl transacetylase component (E2p) of PDHC. Moreover, the experiments show that the N-terminal region of E1p forms an independent folding domain that functions as a binding domain. CD measurements, two-dimensional (2D) (1)H NMR analysis, and secondary structure prediction all indicate that Nterm-E1p has a high alpha-helical content. Here a structural model of the N-terminal domain is proposed. The peptide is present in two conformations, the population of which depends on the sample conditions. The conformations are designated "unfolded" at pH > or =6 and "folded" at pH <5. The 2D (1)H TOCSY spectrum of a mixture of folded and unfolded Nterm-E1p shows exchange cross-peaks that "link" the folded and unfolded state of Nterm-E1p. The rate of exchange between the two species is in the range of 0.5-5 s(-1). Sharp resonances in the NMR spectra of wild-type E1p demonstrate that this 200 kDa enzyme contains highly flexible regions. The observed dynamic character of E1p and of Nterm-E1p is likely required for the binding of the E1p dimer to the two different binding sites on E2p. Moreover, the flexibility might be essential in sustaining the allosteric properties of the enzyme bound in the complex.     

Ethylene emission and responsiveness to applied ethylene vary among Poa species that inherently differ in leaf elongation rates

A plant's ability to produce and respond to ethylene is essential for its vegetative growth. We studied whole-shoot ethylene emission and leaf growth responses to applied ethylene in four Poa spp. that differ inherently in leaf elongation rate and whole-plant relative growth rate. Compared with the fast-growing Poa annua and Poa trivialis, the shoots of the slow-growing species Poa alpina and Poa compressa emitted daily 30% to 50% less ethylene, and their leaf elongation rate was more strongly inhibited when ethylene concentration was increased up to 1 microL L(-1). To our surprise, however, low ethylene concentrations (0.02-0.03 microL L(-1)) promoted leaf growth in the two slow-growing species; at the same concentrations, leaf elongation rate of the two fast-growing species was only slightly inhibited. All responses were observed within 20 min after ethylene applications. Although ethylene generally inhibits growth, our results show that in some species, it may actually stimulate growth. Moreover, in the two slow-growing Poa spp., both growth stimulation and inhibition occurred in a narrow ethylene concentration range, and this effect was associated with a much lower ethylene emission. These findings suggest that the regulation of ethylene production rates and perception of the gas may be more crucial during leaf expansion of these species under non-stressful conditions and that endogenous ethylene concentrations are not large enough to saturate leaf growth responses. In the two fast-growing species, a comparatively higher ethylene endogenous concentration may conversely be present and sufficiently high to saturate leaf elongation responses, invariably leading to growth inhibition.     

Persistent High IgG Phase I Antibody Levels against Coxiella burnetii among Veterinarians Compared to Patients Previously Diagnosed with Acute Q Fever after Three Years of Follow-Up

Background

Little is known about the development of chronic Q fever in occupational risk groups. The aim of this study was to perform long-term follow-up of Coxiella burnetii seropositive veterinarians and investigate the course of IgG phase I and phase II antibodies against C. burnetii antigens and to compare this course with that in patients previously diagnosed with acute Q fever.

Methods

Veterinarians with IgG phase I ≥1:256 (immunofluorescence assay) that participated in a previous seroprevalence study were asked to provide a second blood sample three years later. IgG antibody profiles were compared to a group of acute Q fever patients who had IgG phase I ≥1:256 twelve months after diagnosis.

Results

IgG phase I was detected in all veterinarians (n = 76) and in 85% of Q fever patients (n = 98) after three years (p<0.001). IgG phase I ≥1:1,024, indicating possible chronic Q fever, was found in 36% of veterinarians and 12% of patients (OR 3.95, 95% CI: 1.84–8.49).

Conclusions

IgG phase I persists among veterinarians presumably because of continuous exposure to C. burnetii during their work. Serological and clinical follow-up of occupationally exposed risk groups should be considered.