Closed system O2 uptake: a simple technique of nodule oxygen permeability estimation

Regulation of nitrogen fixation in response to various environmentalconditions often involves an adjustment in nodule permeabilityand, because of the importance of nodule permeability in nitrogenfixation, several methods to estimate it have been developed.In the present study, these methods are reviewed and their limitationsare highlighted. A simple, rapid and inexpensive technique thatcan be used to estimate permeability of nodules and respirationof other plant tissues is described. The technique was evaluatedby comparing it to the lag-phase technique as an independentand reliable method for estimating nodule permeability. Overa wide range of nodule permeability estimates, the closed systemO₂ uptake technique was linearly related to lag-phase permeabilityestimates. The technique was tested further by studying theresponse to sub- and supra-ambient pO₂ in the root environment,and the responses agreed well with published reports on theeffect of O² on nodule permeability. The technique was foundto be very satisfactory in estimating nodule permeability andmay be used to measure the ability of other plant tissues totake up O². Key words: Soybean, Glycine max, nitrogen fixation, root, respiration     

Endoglin is a novel endothelial cell specification gene

Endothelial cells (EC) are important in vasculogenesis and organogenesis during development and in the pathogenesis of cancer and cardiovascular diseases. However, few EC specification factors are known and primary EC production remains inefficient. Based on recent studies implicating endoglin (Eng) in early vascular development and angiogenesis, we hypothesized that Eng may be an EC specification gene. Mouse embryonic stem cells (ESC) were treated with recombinant Eng or a plasmid expressing the Eng ORF, and differentiated in the presence or absence of bone morphogenic protein 4 (BMP4). Expression of the mesoderm and EC marker genes, the known mediators of EC specification and their downstream targets was monitored by quantitative PCR, western blot, immunocytochemistry, and flow cytometry. Functionality of the differentiated EC was assessed by in vitro angiogenesis assay and the induction of Icam1 expression in response to TNF-α treatment. Both recombinant Eng and forced Eng expression increased the number of functional EC expressing the EC marker genes VE-cadherin, vWF, and Tie2, and enhanced the effect of BMP4. The Eng-induced EC differentiation was independent of known mediators of EC specification such as Indian Hedgehog (IHH) and BMP4 or of BMP4/Smad1/5/8 signaling. These studies suggest that Eng is a novel EC specification gene.     

Occurrence,diversity and distribution of Trypanosoma infections in cattle around the Akagera National Park,Rwanda

BackgroundAfrican Trypanosomiases threaten the life of both humans and animals. Trypanosomes are transmitted by tsetse and other biting flies. In Rwanda, the African Animal Trypanosomiasis (AAT) endemic area is mainly around the tsetse-infested Akagera National Park (NP). The study aimed to identify Trypanosoma species circulating in cattle, their genetic diversity and distribution around the Akagera NP.MethodologyA cross-sectional study was carried out in four districts, where 1,037 cattle blood samples were collected. The presence of trypanosomes was determined by microscopy, immunological rapid test VerY Diag and PCR coupled with High-Resolution Melt (HRM) analysis. A parametric test (ANOVA) was used to compare the mean Packed cell Volume (PCV) and trypanosomes occurrence. The Cohen Kappa test was used to compare the level of agreement between the diagnostic methods.FindingsThe overall prevalence of trypanosome infections was 5.6%, 7.1% and 18.7% by thin smear, Buffy coat technique and PCR/HRM respectively. Microscopy showed a low sensitivity while a low specificity was shown by the rapid test (VerY Diag). Trypanosoma (T.) congolense was found at a prevalence of 10.7%, T. vivax 5.2%, T. brucei brucei 2% and T. evansi 0.7% by PCR/HRM. This is the first report of T.evansi in cattle in Rwanda. The non-pathogenic T. theileri was also detected. Lower trypanosome infections were observed in Ankole x Friesian breeds than indigenous Ankole. No human-infective T. brucei rhodesiense was detected. There was no significant difference between the mean PCV of infected and non-infected animals (p>0.162).ConclusionsOur study sheds light on the species of animal infective trypanosomes around the Akagera NP, including both pathogenic and non-pathogenic trypanosomes. The PCV estimation is not always an indication of trypanosome infection and the mechanical transmission should not be overlooked. The study confirms that the area around the Akagera NP is affected by AAT, and should, therefore, be targeted by the control activities. AAT impact assessment on cattle production and information on the use of trypanocides are needed to help policymakers prioritise target areas and optimize intervention strategies. Ultimately, these studies will allow Rwanda to advance in the Progressive Control Pathway (PCP) to reduce or eliminate the burden of AAT.     

The feedback mechanism of nitrate inhibition of nitrogenase activity in soybean may involve asparagine and/or products of its metabolism

A feedback mechanism which involves sensing of change in phloem N concentration has been proposed to control nodulation and dinitrogen fixation in the presence of external combined N. Whether this control is in response to a change in total N or in some specific signal compound(s) is not known. In the present study we reevaluated the hypothesis that control of nodulation and N₂ fixation involves sensing of change in tissue N composition and attempted to identify potential signal molecule(s) involved. Two soybean (Glycine max [L.] Merr.) genotypes (Williams 82 and NOD1-3) differing in nodule number and tolerance to nitrate were germinated in sand trays. Seven-day-old seedlings were inoculated with a solution of Bradyrhizobium japonicum and grown for 28 days in growth chambers, using a hydroponic system with limited N supply to promote nodulation. Half of 28-day-old plants were treated with 15 mM NO₃^?, then control and treated plants were sampled at the onset of nitrogenase inhibition (24 h following NO₃^?, treatment) for evaluation of nitrogenase activity and tissue concentration of total N and of each individual free amino acid. Phenylisothiocyanate-(PITC) amino acid derivatives were separated and quantified using HPLC. The decline in nitrogenase activity following the short-term nitrate treatment was associated with a dramatic asparagine concentration increase in the shoot and an increase in nodule aspartate and glutamate in both genotypes. Asparagine concentration in the shoot increased 35 times from a barely detectable level of 95 to 3 327 nmol g^?1 fresh weight in Williams 82, and more than tripled from 509 to 1 753 nmol g^?1 fresh weight in NOD1-3. Increase in levels of free Asn and in total free amino acids in the shoot following the short-term nitrate treatment was more pronounced in Williams 82 than in its partially nitrate-tolerant mutant NOD1-3. These results indicate that the feedback control of nodule activity may involve sensing changes in shoot asparagine levels and/or products of its metabolism (aspartate and glutamate) in the nodule. These results also indicate that partial-nitrate tolerance of nodulation in the hypernodulated NOD1-3 mutant is associated with a lesser change in tissue N following nitrate treatment.     

Heme-Mediated Induction of CXCL10 and Depletion of CD34+ Progenitor Cells Is Toll-Like Receptor 4 Dependent

Plasmodium falciparum infection can cause microvascular dysfunction, cerebral encephalopathy and death if untreated. We have previously shown that high concentrations of free heme, and C-X-C motif chemokine 10 (CXCL10) in sera of malaria patients induce apoptosis in microvascular endothelial and neuronal cells contributing to vascular dysfunction, blood-brain barrier (BBB) damage and mortality. Endothelial progenitor cells (EPC) are microvascular endothelial cell precursors partly responsible for repair and regeneration of damaged BBB endothelium. Studies have shown that EPC’s are depleted in severe malaria patients, but the mechanisms mediating this phenomenon are unknown. Toll-like receptors recognize a wide variety of pathogen-associated molecular patterns generated by pathogens such as bacteria and parasites. We tested the hypothesis that EPC depletion during malaria pathogenesis is a function of heme-induced apoptosis mediated by CXCL10 induction and toll-like receptor (TLR) activation. Heme and CXCL10 concentrations in plasma obtained from malaria patients were elevated compared with non-malaria subjects. EPC numbers were significantly decreased in malaria patients (P < 0.02) and TLR4 expression was significantly elevated in vivo. These findings were confirmed in EPC precursors in vitro; where it was determined that heme-induced apoptosis and CXCL10 expression was TLR4-mediated. We conclude that increased serum heme mediates depletion of EPC during malaria pathogenesis.