Effects of oxygen and chloramphenicol on Frankia nitrogenase activity

The kinetics of asymbiotic nitrogenase activity in three strains of the actinomycete Frankia were studied. Decay rates for enzyme activity were determined by adding chloramphenicol to active acetylene-reducing cells and measuring the time required for all activity to cease. Synthesis rates were measured by bubbling oxygen through actively-reducing cells (which totally destroyed all activity) and then measuring the time required for activity to return to normal. Decay rates (t _1/2) for these three strains were approximately 30 to 40 min. Synthesis rates were slower and initial nitrogenase activities were recorded about 110 min (DDB 011610) or 210 min (DDB 020210 and WgCc1.17) after return to air-equilibrated cultures. Frankia strain WgCc1.17 showed a greater sensitivity to oxygen and nitrogenase activity was totally lost when cells were bubbled only with atmospheric concentrations of oxygen. The results presented here indicate that nitrogenase activity turnover time is relatively rapid, on the order of minutes rather than hours or days. However, regulation of nitrogenase activity will differ from one strain to another and asmmbiotic characterization will be useful for understanding nitrogenase regulation in the bacterial-plant symbiosis.Contribution no. 879 from the Battelle-Kettering Laboratory     

Genetic and biochemical evidence for involvement of HOTHEAD in the biosynthesis of long-chain α-,ω-dicarboxylic fatty acids and formation of extracellular matrix

In plants, extracellular matrix polymers built from polysaccharides and cuticular lipids have structural and protective functions. The cuticle is found to be ten times thinner in Arabidopsis thaliana (L.) Heynh than in many other plants, and there is evidence that it is unusual in having a high content of α-,ω-dicarboxylic fatty acids (FAs) in its polyesters. We designated the new organ fusion mutant hth-12 after it appeared to be allelic to adhesion of calyx edges (ace) and hothead (hth), upon molecular cloning of the gene by transposon tagging. This mutant is deficient in its ability to oxidize long-chain ω-hydroxy FAs to ω-oxo FAs, which results in leaf polyesters in decreased α-,ω-dicarboxylic FAs and increased ω-hydroxy FAs. These chemical phenotypes lead to disorder of the cuticle membrane structure in hth-12. ACE/HTH is a single-domain protein showing sequence similarity to long-chain FA ω-alcohol dehydrogenases from Candida species, and we hypothesize that it may catalyze the next step after cytochrome P450 FA ω-hydroxylases in the ω-oxidation pathway. We show that ACE/HTH is specifically expressed in epidermal cells. It appears very likely therefore that the changes in the amount of α-,ω-dicarboxylic FAs in hth-12 reflect the different composition of cuticular polyesters. The ACE/HTH gene is also expressed in root epidermal cells which do not form a polyester membrane on the exterior surface, thereby making it possible that the end products of the pathway, α-,ω-dicarboxylic FAs, are generally required for the cross-linking that ensures the integrity of the outer epidermal cell wall.     

Choline stimulates synthesis of extracellular proteins in Trichoderma reesei QM 9414

A correlation between intracellular phospholipid levels and the rate of exoprotein synthesis was investigated in the filamentous fungus Trichoderma reesei QM 9414 during growth on cellulose. When the incubation temperature was varied between 20 and 37°C, the exoprotein synthesis rate correlated with the total cellular amount of phospholipids, but not with an individual phospholipid component. In contrast, when phospholipid bases were added exogenuously, a significant stimulation of exoprotein synthesis was observed with choline. The addition of the surfactant Tween 80—which also stimulates exoprotein secretion in T. reesei QM 9414—prevented choline stimulation. Optimal stimulation occurred around 20 mM choline. Choline stimulated exoprotein synthesis in general as shown by increased activities of several extracellular enzymes. Mycelia required preincubation for at least 20 h before stimulation of choline could be seen. Mycelia pregrown in the absence or presence of choline were equally effective in formation of -glucosidase upon induction with methyl--d-glucoside, and the addition of choline to the induction medium had no effect. Choline did not alter the osmotic stability of protoplasts of T. reesei. Electron microscopic examinations and analysis of chemical constituents as well as marker enzymes from choline grown and non-choline grown mycelia revealed higher contents of mitochondria and endoplasmic reticula in choline grown mycelia. The possibility is discussed that choline may stimulate exoprotein synthesis by increasing the cellular content of endoplasmic reticula.     

Nitrogen in shoot litter,root litter and root exudates from nitrogen-fixingAlnus incana

Summary A pot experiment withAlnus incana (L.) Moench growing in sand was set up to compare the amounts of nitrogen released from plants shoot litter with that released below ground as root litter and/or root exudation. No nitrogen fixation by free-living microorganisms was found in the sand and the increased nitrogen content of the plant + soil system was therefore due to nitrogen fixation byFrankia in the alder root-nodules. Most of the nitrogen released from the plants was in the nitrogen-rich leaf and other shoot litter. Only small amounts of nitrogen were found in the drainage water from the pots and were recorded as increased nitrogen content of the sand.     

Biochemical engineering of the N-acyl side chain of sialic acid leads to increased calcium influx from intracellular compartments and promotes differentiation of HL60 cells

Sialylation of glycoconjugates is essential for mammalian cells. Sialic acid is synthesized in the cytosol from N-acetylmannosamine by several consecutive steps. Using N-propanoylmannosamine, a novel precursor of sialic acid, we are able to incorporate unnatural sialic acids with a prolonged N-acyl side chain (e.g., N-propanoylneuraminic acid) into glycoconjugates taking advance of the cellular sialylation machinery. Here, we report that unnatural sialylation of HL60-cells leads to an increased release of intracellular calcium after application of thapsigargin, an inhibitor of SERCA Ca2+-ATPases. Furthermore, this increased intracellular calcium concentration leads to an increased adhesion to fibronectin. Finally, we observed an increase of the lectin galectin-3, a marker of monocytic differentiation of HL60-cells.     

The effect of hexose monophosphate shunt inhibitors on adenohypophyseal and ceruloplasmin reactions to oestrogen.

Oestradiol benzoate, as an aqueous microcrystal suspension, was administered i.m. to rats in doses of 1 mg twice a week; it induced adenohypophyseal hyperplasia and an increase of the thyroxine-binding capacity of the adenohypophyseal proteins in vitro and raised the blood ceruloplasmin level. The simultaneous administration of a hexose monophosphate shunt inhibitor--6-aminonicotinamide (200 microgram/rat/day in food) or oxythiamine (8 mg/rat/day in food)--did not modify the reaction of the adenohypophysis; the hexose monophosphate shunt thus probably does not play a significant role in the adenohypophyseal reaction to oestrogens. By themselves, both inhibitors raised the blood ceruloplasmin level and their effect summated with that of oestradiol. The mechanism of action of the inhibitors is not known, but a nonspecific stress effect leading to an increase in the ceruloplasmin level as an "acute phase protein" is considered to be the most likely.     

Dendritic Cells in Chronic Mycobacterial Granulomas Restrict Local Anti-Bacterial T Cell Response in a Murine Model

Background

Mycobacterium-induced granulomas are the interface between bacteria and host immune response. During acute infection dendritic cells (DCs) are critical for mycobacterial dissemination and activation of protective T cells. However, their role during chronic infection in the granuloma is poorly understood.

Methodology/Principal Findings

We report that an inflammatory subset of murine DCs are present in granulomas induced by Mycobacteria bovis strain Bacillus Calmette-guerin (BCG), and both their location in granulomas and costimulatory molecule expression changes throughout infection. By flow cytometric analysis, we found that CD11c⁺ cells in chronic granulomas had lower expression of MHCII and co-stimulatory molecules CD40, CD80 and CD86, and higher expression of inhibitory molecules PD-L1 and PD-L2 compared to CD11c⁺ cells from acute granulomas. As a consequence of their phenotype, CD11c⁺ cells from chronic lesions were unable to support the reactivation of newly-recruited, antigen 85B-specific CD4⁺IFNγ⁺ T cells or induce an IFNγ response from naïve T cells in vivo and ex vivo. The mechanism of this inhibition involves the PD-1:PD-L signaling pathway, as ex vivo blockade of PD-L1 and PD-L2 restored the ability of isolated CD11c⁺ cells from chronic lesions to stimulate a protective IFNγ T cell response.

Conclusions/Significance

Our data suggest that DCs in chronic lesions may facilitate latent infection by down-regulating protective T cell responses, ultimately acting as a shield that promotes mycobacterium survival. This DC shield may explain why mycobacteria are adapted for long-term survival in granulomatous lesions.     

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.     

Serological Evidence of Chikungunya Virus among Acute Febrile Patients in Southern Mozambique

Background

In the last two decades, chikungunya virus (CHIKV) has rapidly expanded to several geographical areas, causing frequent outbreaks in sub-Saharan Africa, South East Asia, South America, and Europe. Therefore, the disease remains heavily neglected in Mozambique, and no recent study has been conducted.

Methods

Between January and September 2013, acute febrile patients with no other evident cause of fever and attending a health center in a suburban area of Maputo city, Mozambique, were consecutively invited to participate. Paired acute and convalescent serum samples were requested from each participant. Convalescent samples were initially screened for anti-CHIKV IgG using a commercial indirect immunofluorescence test, and if positive, the corresponding acute sample was screened using the same test.

Results

Four hundred patients were enrolled. The median age of study participants was 26 years (IQR: 21–33 years) and 57.5% (224/391) were female. Paired blood samples were obtained from 209 patients, of which 26.4% (55/208) were presented anti-CHIKV IgG antibodies in the convalescent sample. Seroconversion or a four-fold titer rise was confirmed in 9 (4.3%) patients.

Conclusion

The results of this study strongly suggest that CHIKV is circulating in southern Mozambique. We recommend that CHIKV should be considered in the differential diagnosis of acute febrile illness in Mozambique and that systematic surveillance for CHIKV should be implemented.