Effects of diffusion limitation on immobilized nitrifying microorganisms at low temperatures

Activation energies of suspended and immobilized nitrifying bacteria were determined and compared to determine if diffusion limitation results in decreased sensitivity for temperature. The activation energy for the respiration activity of suspended Nitrosomonas europaea and Nitrobacter agilis was found to be 86.4 and 58.4 kJ mol(-1), respectively. The activation energy for oxygen diffusion in the support material, kappa-carrageenan, determined from the effect of temperature on the effective diffusion coefficient (D), was 17.2 kJ mol(-1). Consequently, the apparent actvation energy of diffusion limited cells should be lower. It was indeed shown that due to the effect of diffusion limitation and to temperature effects on the Monod constant K(s), the immobilized-cell activity was less sensitive to temperature. The apparent activation energy for immobilized Ns. europaea was between 28.6 and 94.2 kJ mol(-1) and for immobilized Nb. agilis between 1.4 and 72.9 kJ mol(-1), depending on the oxygen concentration and temperature. (c) 1995 John Wiley & Sons, Inc.     

The enzymes of the ammonia assimilation in Pseudomonas aeruginosa

Glutamine synthetase from Pseudomonas aeruginosa is regulated by repression/derepression of enzyme synthesis and by adenylylation/deadenylylation control. High levels of deadenylylated biosynthetically active glutamine synthetase were observed in cultures growing with limiting amounts of nitrogen while synthesis of the enzyme was repressed and that present was adenylylated in cultures with excess nitrogen.NADP-and NAD-dependent glutamate dehydrogenase could be separated by column chromatography and showed molecular weights of 110,000 and 220,000, respectively. Synthesis of the NADP-dependent glutamate dehydrogenase is repressed under nitrogen limitation and by growth on glutamate. In contrast, NAD-dependent glutamate dehydrogenase is derepressed by glutamate. Glutamate synthase is repressed by glutamate but not by excess nitrogen.     

Floristic variation and willow carr development within a southwest England wetland

Abstract. Woodland colonization on wetlands is considered to have a detrimental effect on their ecological value, even though detailed analysis of this process is lacking. This paper provides an evaluation of the ecological changes resulting from succession of poor fen (base‐poor mire) to willow wet woodland on Goss Moor NNR in Cornwall, UK. Different ages of willow carr were associated with eight understorey communities. During willow colonization, in the ground flora, there was a progressive decrease in poor fen species and an associated increase in woodland species, which appeared to be related to an increase in canopy cover and therefore shade. The most diverse community was found to be the most recent willow and was dominated by poor fen species. The oldest willow was the second most diverse and was associated with a reduction in poor fen species and an increase in woodland species. Architectural features were used successfully to assess the general condition and structure of willow. Tree height and DBH were identified as useful parameters to accurately assess willow age in the field. The implications of active intervention to remove willow in order to conserve the full range of communities within the hydrosere are discussed.     

Effect of consumption of phenols from olives and extra virgin olive oil on LDL oxidizability in healthy humans

A high intake of olive oil has been proposed as an explanation for the low incidence of coronary heart disease in Mediterranean countries, but it is unclear whether olive oil offers specific benefits beyond a low content of saturated fat. Some types of extra virgin olive oil are rich in non-polar phenols, which might be taken up by plasma LDL particles and protect these from becoming atherogenic by oxidative modification. In a pilot study we found that consumption of 47 g fortified olive oil containing 31 mg phenols significantly increased the lag time of LDL oxidation from 112 ± 5 min before to 130 ± 7 min 2h after the meal. However, this study was not controlled, and in the current study we therefore investigated whether olive oil phenols increase the lag time of LDL oxidation in postprandial samples when compared with a control group.

Twelve healthy men and women consumed four different olive oil supplements with a meal on four separate occasions: one similar to the supplement in the pilot study (positive control); one containing mainly non-polar olive oil phenols; one containing mainly polar olive oil phenols; and one without phenols (placebo). Lag time significantly increased 2 h after the meals with the positive control (8 ± 2 min), the polar phenols (8 ± 2 min), and the placebo (8 ± 2 min), but not after the non-polar phenols (-0.4 ± 3 min). Increases were not statistically different between supplements.

These results indicate that the lag time of LDL-oxidation is increased after consumption of a meal. This increase is probably due to non-specific meal or time effects and not to phenols from olives or olive oil. Furthermore, these findings stress the need for adequate controlled studies to avoid misinterpretations of the data.     

A Shift towards Pro-Inflammatory CD16+ Monocyte Subsets with Preserved Cytokine Production Potential after Kidney Transplantation

Background

The presence of monocyte-macrophage lineage cells in rejecting kidney transplants is associated with worse graft outcome. At present, it is still unclear how the monocyte-macrophage related responses develop after transplantation. Here, we studied the dynamics, phenotypic and functional characteristics of circulating monocytes during the first 6 months after transplantation and aimed to establish the differences between kidney transplant recipients and healthy individuals.

Methods

Phenotype, activation status and cytokine production capacity of classical (CD14++CD16−), intermediate (CD14++CD16+) and non-classical (CD14+CD16++), monocytes were determined by flow cytometry in a cohort of 33 healthy individuals, 30 renal transplant recipients at transplantation, 19 recipients at 3 months and 16 recipients at 6 months after transplantation using a cross-sectional approach.

Results

The percentage of both CD16+ monocyte subsets was significantly increased in transplant recipients compared to healthy individuals, indicative of triggered innate immunity (p≤0.039). Enhanced production capacity of tumor necrosis factor-α, interferon-γ and interleukin-1β was observed by monocytes at transplantation compared to healthy individuals. Remarkably, three months post-transplant, in presence of potent immunosuppressive drugs and despite improved kidney function, interferon-γ, tumor necrosis factor-α and interleukin-10 production capacity still remained significantly increased.

Conclusion

Our data demonstrate a skewed balance towards pro-inflammatory CD16+ monocytes that is present at the time of transplantation and retained for at least 6 months after transplantation. This shift could be one of the important drivers of early post-transplant cellular immunity.     

Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage

CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids.

A major downside of double-strand break-mediated genome editing is the need to verify the genomic integrity of the targeted locus and confirm the absence of off-target indels. This study shows that in-trans paired nicking is a mutation-free CRISPR strategy to introduce precise knock-ins into human organoids; its genomic fidelity allows all knock-in cells to be pooled, accelerating the establishment of new organoid models.     

Stimulation of Na+,K+-ATPase activity, increase in potassium uptake, and enhanced production of ouabain-like compounds in ammonia-treated mouse astrocytes

Active potassium (K+) uptake and Na+,K+-ATPase activity were measured in primary cultures of mouse astrocytes. Both parameters were virtually unaffected by acute ammonia treatment but increased after chronic exposure to pathophysiologically relevant concentrations of ammonia (0.3 or 3 mM) for 1-4 days. The increased Na+,K+-ATPase activity after chronic treatment with ammonia was further enhanced in the acute presence of 12 mM K+. Based on these observations and literature data it was hypothesized that the direct effect of ammonia is formation of easily diffusible compound(s) with ouabain-like effect, that upregulation occurs of Na+,K+-ATPase activity and K+ uptake in response to the resulting ATPase inhibition, and that the washing procedure preceding the uptake experiments and the determination of Na+,K+-ATPase activity unmasks the upregulation. To test this hypothesis, the content of compounds with ouabain-like action was measured in media in which astrocytes had been incubated in the presence of 3 mM ammonia for 4 days and in controls to which an additional 3 mM NaCl had been added instead of ammonia. An endogenous, compound with ouabain-like activity was demonstrated both under control conditions and in the ammonia-treated cultures, and the content of this compound was increased by 50% in the ammonia-treated cultures. Preliminary experiments showed that at least part of the released ouabain-like compounds cross-react with authentic ouabain.     

Chronic blockade of brain "ouabain" prevents sympathetic hyper-reactivity and impairment of acute baroreflex resetting in rats with congestive heart failure

In rats with congestive heart failure (CHF) post myocardial infarction (MI) acute blockade of brain "ouabain" reverses sympathetic hyperactivity and chronic blockade prevents the desensitization of baroreflex function. This study was conducted to determine: i) if chronic blockade of brain "ouabain" maintains normal sympathetic reactivity; and ii) if acute baroreflex resetting (another parameter of baroreflex function) also becomes impaired, and if so, does brain "ouabain" contribute to impairment in acute baroreflex resetting. CHF post MI was induced by acute coronary artery ligation in Wistar rats. Animals were treated with 200 microg x day(-1) i.c.v. or i.v. Fab fragments (which bind brain "ouabain" with high affinity), or treated with 200 microg x day(-1) i.c.v. gamma-globulins (control group). The length of treatment was 0.5-8 weeks or 4-8 weeks post MI. At 8 weeks mean arterial pressure (MAP), central venous pressure (CVP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were recorded in concious rats at rest and in response to: i) air-jet stress, ii) i.c.v. guanabenz (an alpha2-adrenoceptor agonist), and iii) a 30 min i.v. infusion of nitroprusside (NP). Excitatory responses to air stress and inhibitory responses to guanabenz of MAP, HR, and RSNA were significantly enhanced in rats with CHF versus the sham-operated treated group. This enhancement was prevented in the CHF group treated with i.c.v., but not i.v., Fab. Nitroprusside induced a sustained decrease in MAP (approximately 25 mmHg) and a transient decrease in CVP. Heart rate and RSNA increased significantly within 1 min of beginning the infusion. The peak increases as well as the product of changes in MAP-HR and RSNA-HR were significantly smaller in rats with CHF treated with gamma-globulins versus sham rats and versus CHF rats treated with i.c.v. Fab. In sham-operated rats and CHF rats treated with i.c.v. Fab, RSNA and HR began to decrease within 3-4 min of beginning the NP infusion and had returned to baseline by 20 min. In contrast, RSNA and HR remained increased throughout the infusion in the CHF rats treated with gamma-globulins. These data indicate that in rats with CHF acute resetting of the arterial baroreflex in response to a lower BP becomes impaired, and chronic blockade of brain "ouabain" prevents both this change in baroreflex resetting as well as sympathetic hyperactivity.