Dimethylarginine Dimethylaminohydrolase-1 Transgenic Mice Are Not Protected from Ischemic Stroke

Background

Methylated arginines are endogenous analogues of L-arginine, the substrate for nitric oxide (NO) synthase. Asymmetric dimethylarginine (ADMA) interferes with NO formation, causing endothelial dysfunction. ADMA is a predictor of cardiovascular events and mortality in humans. It is eliminated primarily by enzymatic activity of dimethylarginine dimethylaminohydrolase (DDAH).

Methodology/Principal Findings

We investigated whether human DDAH-1 (hDDAH-1) transgenicity protects from ischemic tissue damage in temporary middle cerebral artery occlusion (tMCAO) in mice. Infarct sizes did not significantly differ between hDDAH-1 transgenic (TG) mice and wild-type littermates (WT). As expected, ADMA plasma concentrations were significantly decreased, cerebral hDDAH expression and protein significantly increased in transgenic animals. Interestingly, neither brain tissue DDAH activity nor ADMA concentrations were different between TG and WT mice. In contrast, muscular DDAH activity was generally lower than in brain but significantly increased in TG mice.

Conclusion/Significance

Our study demonstrates that hDDAH-1 transgenic mice are not protected from ischemic cerebral tissue damage in tMCAO. This lack of protection is due to high basal cerebral DDAH activity, which is not further increasable by transgenic overexpression of DDAH.     

Evaluation of different glycoforms of honeybee venom major allergen phospholipase A2 (Api m 1) produced in insect cells

Allergic reactions to hymenoptera stings are one of the major reasons for IgE-mediated anaphylaxis. However, proper diagnosis using venom extracts is severely affected by molecular cross-reactivity. In this study recombinant honeybee venom major allergen phospholipase A2 (Api m 1) was produced for the first time in insect cells. Using baculovirus infection of different insect cell lines allergen versions providing a varying degree of cross-reactive carbohydrate determinants as well as a non glycosylated variant could be obtained as secreted soluble proteins in high yields. The resulting molecules were analyzed for their glycosylation and proved to show advantageous properties regarding cross-reactivity in sIgE-based assays. Additionally, in contrast to the enzymatically active native protein the inactivated allergen did not induce IgE-independent effector cell activation. Thus, insect cell-derived recombinant Api m 1 with defined CCD phenotypes might provide further insights into hymenoptera venom IgE reactivities and contribute to an improved diagnosis of hymenoptera venom allergy.     

Substrate recognition by complement convertases revealed in the C5-cobra venom factor complex

Complement acts as a danger‐sensing system in the innate immune system, and its activation initiates a strong inflammatory response and cleavage of the proteins C3 and C5 by proteolytic enzymes, the convertases. These contain a non‐catalytic substrate contacting subunit (C3b or C4b) in complex with a protease subunit (Bb or C2a). We determined the crystal structures of the C3b homologue cobra venom factor (CVF) in complex with C5, and in complex with C5 and the inhibitor SSL7 at 4.3 Å resolution. The structures reveal a parallel two‐point attachment between C5 and CVF, where the presence of SSL7 only slightly affects the C5–CVF interface, explaining the IgA dependence for SSL7‐mediated inhibition of C5 cleavage. CVF functions as a relatively rigid binding scaffold inducing a conformational change in C5, which positions its cleavage site in proximity to the serine protease Bb. A general model for substrate recognition by the convertases is presented based on the C5–CVF and C3b–Bb–SCIN structures. Prior knowledge concerning interactions between the endogenous convertases and their substrates is rationalized by this model.     

Structural olfactory nerve changes in patients suffering from idiopathic intracranial hypertension

Background

Complications of idiopathic intracranial hypertension (IIH) are usually caused by elevated intracranial pressure (ICP). In a similar way as in the optic nerve, elevated ICP could also compromise the olfactory nerve system. On the other side, there is growing evidence that an extensive lymphatic network system around the olfactory nerves could be disturbed in cerebrospinal fluid disorders like IIH. The hypothesis that patients with IIH suffer from hyposmia has been suggested in the past. However, this has not been proven in clinical studies yet. This pilot study investigates whether structural changes of the olfactory nerve system can be detected in patients with IIH.

Methodology/Principal Findings

Twenty-three patients with IIH and 23 matched controls were included. Olfactory bulb volume (OBV) and sulcus olfactorius (OS) depth were calculated by magnetic resonance techniques. While mean values of total OBV (128.7±38.4 vs. 130.0±32.6 mm³, p=0.90) and mean OS depth (8.5±1.2 vs. 8.6±1.1 mm, p=0.91) were similar in both groups, Pearson correlation showed that patients with a shorter medical history IIH revealed a smaller OBV (r=0.53, p<0.01). In untreated symptomatic patients (n=7), the effect was greater (r=0.76, p<0.05). Patients who suffered from IIH for less than one year (n=8), total OBV was significantly smaller than in matched controls (116.6±24.3 vs. 149.3±22.2 mm³, p=0.01). IIH patients with visual disturbances (n=21) revealed a lower OS depth than patients without (8.3±0.9 vs. 10.8±1.0 mm, p<0.01).

Conclusions/Significance

The results suggest that morphological changes of the olfactory nerve system could be present in IIH patients at an early stage of disease.     

The influence of elevated atmospheric CO2 on fine root dynamics in an intact temperate forest

Root dynamics are important for plant, ecosystem and global carbon cycling. Changes in root dynamics caused by rising atmospheric CO₂ not only have the potential to moderate further CO₂ increases, but will likely affect forest function. We used FACE (Free‐Air CO₂ Enrichment) to expose three 30‐m diameter plots in a 13‐year‐old loblolly pine (Pinus taeda) forest to elevated (ambient + 200 µL L^?1) atmospheric CO₂. Three identical fully instrumented plots were implemented as controls (ambient air only). We quantified root dynamics from October 1998 to October 1999 using minirhizotrons. In spite of 16% greater root lengths and 24% more roots per minirhizotron tube, the effects of elevated atmospheric CO₂ on root lengths and numbers were not statistically significant. Similarly, production and mortality were also unaffected by the CO₂ treatment, even though annual root production and mortality were 26% and 46% greater in elevated compared to ambient CO₂ plots. Average diameters of live roots present at the shallowest soil depth were, however, significantly enhanced in CO₂‐enriched plots. Mortality decreased with increasing soil depth and the slopes of linear regression lines (mortality vs. depth) differed between elevated and ambient CO₂ treatments, reflecting the significant CO₂ by depth interaction. Relative root turnover (root flux/live root pool) was unchanged by exposure to elevated atmospheric CO₂. Results from this study suggest modest, if any, increases in ecosystem‐level root productivity in CO₂‐enriched environments.     

Population kinetics of homoarginine and optimized supplementation for cardiovascular risk reduction

Homoarginine is an endogenous amino acid whose levels are reduced in patients with renal, cardio- and cerebrovascular disease. Moreover, low homoarginine concentrations independently predict morbidity and mortality in these patients. Besides endogenous synthesis, homoarginine is also a constituent of the human diet. The objective of the present study was to analyze the kinetics of orally supplemented homoarginine in human plasma by means of a pharmacometric approach. We developed a pharmacometric model to evaluate different dosing regimens, especially the regimen of 125 mg once weekly, based on a previous clinical study (n?=?20). The model was adapted to account for differences in baseline homoarginine plasma concentrations between healthy and diseased individuals. A novel dosing regimen of 25 mg once daily led to higher attainment of homoarginine reference concentrations using clinical trial simulations. With 25 mg/day, the trough concentration of only 6% of the older and 3.8% of the younger population was predicted to be below the target concentration of 2.0–4.1 µmol/L. In synopsis, the new dosing regimen recapitulates the kinetics of homoarginine in healthy individuals optimally.

     

Carbon sequestration potential of soils in southeast Germany derived from stable soil organic carbon saturation

Sequestration of atmospheric carbon (C) in soils through improved management of forest and agricultural land is considered to have high potential for global CO₂ mitigation. However, the potential of soils to sequester soil organic carbon (SOC) in a stable form, which is limited by the stabilization of SOC against microbial mineralization, is largely unknown. In this study, we estimated the C sequestration potential of soils in southeast Germany by calculating the potential SOC saturation of silt and clay particles according to Hassink [Plant and Soil 191 (1997) 77] on the basis of 516 soil profiles. The determination of the current SOC content of silt and clay fractions for major soil units and land uses allowed an estimation of the C saturation deficit corresponding to the long‐term C sequestration potential. The results showed that cropland soils have a low level of C saturation of around 50% and could store considerable amounts of additional SOC. A relatively high C sequestration potential was also determined for grassland soils. In contrast, forest soils had a low C sequestration potential as they were almost C saturated. A high proportion of sites with a high degree of apparent oversaturation revealed that in acidic, coarse‐textured soils the relation to silt and clay is not suitable to estimate the stable C saturation. A strong correlation of the C saturation deficit with temperature and precipitation allowed a spatial estimation of the C sequestration potential for Bavaria. In total, about 395 Mt CO₂‐equivalents could theoretically be stored in A horizons of cultivated soils – four times the annual emission of greenhouse gases in Bavaria. Although achieving the entire estimated C storage capacity is unrealistic, improved management of cultivated land could contribute significantly to CO₂ mitigation. Moreover, increasing SOC stocks have additional benefits with respect to enhanced soil fertility and agricultural productivity.     

Basophil Activation Test Using Recombinant Allergens: Highly Specific Diagnostic Method Complementing Routine Tests in Wasp Venom Allergy

Background

Skin testing can expose allergic subjects to potential systemic reactions, sensitization against unrelated proteins, and increased risk of future sting reactions. Therefore the continuous improvement of in vitro diagnostic methods is desirable. Recombinant allergens have been shown to improve the sensitivity of specific IgE (sIgE) detection in vitro whilst no data is available regarding their application and reliability in basophil activation test (BAT). Here we aimed to compare the specificity and sensitivity of recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 in both specific IgE (sIgE) detection in vitro and basophil activation test.

Methods

sIgE detection by ELISA or ImmunoCAP and BAT towards the panel of recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 were performed in 43 wasp venom allergic patients with a history of anaphylactic reaction and sIgE seropositivity, as well as 17 controls defined as subjects with a history of repetitive wasp stings but absence of any allergic symptom.

Results

The BAT performed with the recombinant allergens Ves v 1, Ves v 2, Ves v 3 and Ves v 5 markedly improved the specificity of diagnosis in wasp venom allergic subjects when compared to the respective sIgE detection in serum.

Conclusions

BAT performed with the recombinant allergens Ves v 5, Ves v 3 and Ves v 1 provides an emerging highly specific in vitro method for the detection of wasp venom allergy, compared to the sIgE detection. Recombinant allergens applied to BAT represent a step forward in developing reliable in vitro tests for specific diagnosis of allergy.     

Hypercholesterolemia impairs basal nitric oxide synthase turnover rate: a study investigating the conversion of L-[guanidino-(15)N(2)]-arginine to (15)N-labeled nitrate by gas chromatography--mass spectrometry.

Endothelial function is impaired in hypercholesterolemia and atherosclerosis, which is probably due to reduced biological activity of endothelium-derived nitric oxide (NO). NO is synthesized in functionally intact endothelium by oxidation of the terminal guanidino nitrogen atom(s) of the amino acid precursor, L-arginine. We applied stable isotope dilution techniques and gas chromatographic-mass spectrometric approaches to investigate metabolism of L-[guanidino-(15)N(2)]-arginine to (15)N-labeled nitrate in hypercholesterolemic rabbits and controls. After 4 weeks on control or 1% cholesterol-enriched diet, rabbits received 267 +/- 6 micromol of L-[guanidino-(15)N(2)]-arginine/kg of body weight via gastric cannulation. (15)N-isotope content of L-arginine in plasma and in platelet lysates increased 2h later in both groups, and almost returned to baseline until 24h. (15)N-isotope content of plasma nitrite and nitrate also increased in both groups at 2h, and had almost returned to natural content 24h later. (15)N-isotope content of urinary nitrate was significantly increased in control animals in urines collected from 0 to 12, 12 to 24, and had returned to baseline in the urine sample collected from 24 to 48 h. In the cholesterol group only a slight, insignificant elevation of (15)N-isotope content was observed for urinary nitrate. The extent of conversion of L-[guanidino-(15)N(2)]-arginine to (15)N-labeled nitrate was strongly and inversely correlated to plasma concentration of the endogenous NO synthase inhibitor, asymmetric dimethylarginine (ADMA), which was elevated in cholesterol-fed rabbits (R=0.77; p < 0.05). Our data show that baseline NO synthase turnover rate is reduced in rabbits during early hypercholesterolemia. Our study gives evidence that the mechanism of the impaired conversion of L-[guanidino-(15)N(2)]-arginine to (15)N-labeled nitrate most likely involves inhibition of NO synthase by ADMA, which is present in elevated concentrations in hypercholesterolemia.