Characterization of Early Disease Status in Treatment-Naive Male Paediatric Patients with Fabry Disease Enrolled in a Randomized Clinical Trial

Trial Design

This analysis characterizes the degree of early organ involvement in a cohort of oligo-symptomatic untreated young patients with Fabry disease enrolled in an ongoing randomized, open-label, parallel-group, phase 3B clinical trial.

Methods

Males aged 5–18 years with complete α-galactosidase A deficiency, without symptoms of major organ damage, were enrolled in a phase 3B trial evaluating two doses of agalsidase beta. Baseline disease characteristics of 31 eligible patients (median age 12 years) were studied, including cellular globotriaosylceramide (GL-3) accumulation in skin (n = 31) and kidney biopsy (n = 6; median age 15 years; range 13–17 years), renal function, and glycolipid levels (plasma, urine).

Results

Plasma and urinary GL-3 levels were abnormal in 25 of 30 and 31 of 31 patients, respectively. Plasma lyso-GL-3 was elevated in all patients. GL-3 accumulation was documented in superficial skin capillary endothelial cells (23/31 patients) and deep vessel endothelial cells (23/29 patients). The mean glomerular filtration rate (GFR), measured by plasma disappearance of iohexol, was 118.1 mL/min/1.73 m² (range 90.4–161.0 mL/min/1.73 m²) and the median urinary albumin/creatinine ratio was 10 mg/g (range 4.0–27.0 mg/g). On electron microscopy, renal biopsy revealed GL-3 accumulation in all glomerular cell types (podocytes and parietal, endothelial, and mesangial cells), as well as in peritubular capillary and non-capillary endothelial, interstitial, vascular smooth muscle, and distal tubules/collecting duct cells. Lesions indicative of early Fabry arteriopathy and segmental effacement of podocyte foot processes were found in all 6 patients.

Conclusions

These data reveal that in this small cohort of children with Fabry disease, histological evidence of GL-3 accumulation, and cellular and vascular injury are present in renal tissues at very early stages of the disease, and are noted before onset of microalbuminuria and development of clinically significant renal events (e.g. reduced GFR). These data give additional support to the consideration of early initiation of enzyme replacement therapy, potentially improving long-term outcome.

Trial Registration

ClinicalTrials.gov NCT00701415     

The selenium-rich C-terminal domain of mouse selenoprotein P is necessary for the supply of selenium to brain and testis but not for the maintenance of whole body selenium

Selenoprotein P (Sepp1) has two domains with respect to selenium content: the N-terminal, selenium-poor domain and the C-terminal, selenium-rich domain. To assess domain function, mice with deletion of the C-terminal domain have been produced and compared with Sepp1-/- and Sepp1+/+ mice. All mice studied were males fed a semipurified diet with defined selenium content. The Sepp1 protein in the plasma of mice with the C-terminal domain deleted was determined by mass spectrometry to terminate after serine 239 and thus was designated Sepp1Delta240-361. Plasma Sepp1 and selenium concentrations as well as glutathione peroxidase activity were determined in the three types of mice. Glutathione peroxidase and Sepp1Delta240-361 accounted for over 90% of the selenium in the plasma of Sepp1Delta240-361 mice. Calculations using results from Sepp1+/+ mice revealed that Sepp1, with a potential for containing 10 selenocysteine residues, contained an average of 5 selenium atoms per molecule, indicating that shortened and/or selenium-depleted forms of the protein were present in these wild-type mice. Sepp1Delta240-361 mice had low brain and testis selenium concentrations that were similar to those in Sepp1-/- mice but they better maintained their whole body selenium. Sepp1Delta240-361 mice had depressed fertility, even when they were fed a high selenium diet, and their spermatozoa were defective and morphologically indistinguishable from those of selenium-deficient mice. Neurological dysfunction and death occurred when Sepp1Delta240-361 mice were fed selenium-deficient diet. These phenotypes were similar to those of Sepp1-/- mice but had later onset or were less severe. The results of this study demonstrate that the C terminus of Sepp1 is critical for the maintenance of selenium in brain and testis but not for the maintenance of whole body selenium.     

Accurate quantification of dimethylamine (DMA) in human urine by gas chromatography-mass spectrometry as pentafluorobenzamide derivative: evaluation of the relationship between DMA and its precursor asymmetric dimethylarginine (ADMA) in health and disease

Dimethylamine [DMA, (CH(3))(2)NH)] is abundantly present in human urine. Main sources of urinary DMA have been reported to include trimethylamine N-oxide, a common food component, and asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis. ADMA is excreted in the urine in part unmetabolized and in part after hydrolysis to DMA by dimethylarginine dimethylaminohydrolase (DDAH). Here we describe a GC-MS method for the accurate and rapid quantification of DMA in human urine. The method involves use of (CD(3))(2)NH as internal standard, simultaneous derivatization with pentafluorobenzoyl chloride and extraction in toluene, and selected-ion monitoring of m/z 239 for DMA and m/z 245 for (CD(3))(2)NH in the electron ionization mode. GC-MS analysis of urine samples from 10 healthy volunteers revealed a DMA concentration of 264+/-173 microM equivalent to 10.1+/-1.64 micromol/mmol creatinine. GC-tandem MS analysis of the same urine samples revealed an ADMA concentration of 27.3+/-15.3 microM corresponding to 1.35+/-1.2 micromol/mmol creatinine. In these volunteers, a positive correlation (R=0.83919, P=0.0024) was found between urinary DMA and ADMA, with the DMA/ADMA molar ratio being 10.8+/-6.2. Elevated excretion rates of DMA (52.9+/-18.5 micromol/mmol creatinine) and ADMA (3.85+/-1.65 micromol/mmol creatinine) were found by the method in 49 patients suffering from coronary artery disease, with the DMA/ADMA molar ratio also being elevated (16.8+/-12.8). In 12 patients suffering from end-stage liver disease, excretion rates of DMA (47.8+/-19.7 micromol/mmol creatinine) and ADMA (5.6+/-1.5 micromol/mmol creatinine) were found to be elevated, with the DMA/ADMA molar ratio (9.17+/-4.2) being insignificantly lower (P=0.46). Between urinary DMA and ADMA there was a positive correlation (R=0.6655, P<0.0001) in coronary artery disease, but no correlation (R=0.27339) was found in end-stage liver disease.     

Orphan selenoproteins

Selenoproteins contain selenium in stoichiometric amounts. Most are synthesized by a process that decodes UGA codons as selenocysteine. Twelve animal selenoproteins have been characterized, and biochemical functions have been described for all but three. Two of these “orphan” selenoproteins are discussed in this paper. Selenoprotein P is an extracellular glycoprotein that contains multiple selenocysteines. It binds heparin and associates with endothelial cells. Two isoforms have been identified. Plasma concentration of selenoprotein P correlates with protection against diquat liver injury, suggesting that the protein protects against oxidant injury. Selenoprotein W is a small intracellular protein that contains one selenocysteine. It binds glutathione and has been suggested to function in oxidant defense. The postulated oxidant defense properties of these selenoproteins are consistent with the facile thiol-redox properties of selenocysteine. It can be predicted that more proteins will be discovered that take advantage of the chemical properties of selenium. BioEssays 21:231–237, 1999. © 1999 John Wiley & Sons, Inc.     

Low cortisol levels in blood from dairy cows with ketosis: a field study

Background  

An elevated plasma glucose concentration has been considered to be a potential risk factor in the pathogenesis of left-displaced abomasums (DA). Therefore the present study was performed to investigate if spontaneous disease (parturient paresis, metritis, ketosis etc) in dairy cows results in elevated concentrations of glucose and cortisol in blood as cortisol is the major regulator of glucose in ruminants.     

The Interaction of Equine Lysozyme:Oleic Acid Complexes with Lipid Membranes Suggests a Cargo Off-Loading Mechanism

The normal function of equine lysozyme (EL) is the hydrolysis of peptidoglycan residues of bacterial cell walls. EL is closely related to α-lactalbumins with respect to sequence and structure and further possesses the calcium binding site of α-lactalbumins. Recently, EL multimeric complexes with oleic acids (ELOAs) were shown to possess tinctorial and morphological properties, similar to amyloidal aggregates, and to be cytotoxic. ELOA's interactions with phospholipid membranes appear to be central to its biological action, similar to human α-lactalbumin made lethal to tumor cells. Here, we describe the interaction of ELOA with phospholipid membranes. Confocal scanning laser microscopy shows that ELOA, but not native EL, accumulates on the surface of giant unilamellar vesicles, without inducing significant membrane permeability. Quartz crystal microbalance with dissipation data indicated an essentially non-disruptive binding of ELOA to supported lipid bilayers, leading to formation of highly dissipative and “soft” lipid membrane; at higher concentrations of ELOA, the lipid membrane desorbs from the surface probably as bilayer sheets of vesicles. This membrane rearrangement occurred to a similar extent when free oleic acid (OA) was added, but not when free OA was removed from ELOA by prior incubation with bovine serum albumin, emphasizing the role of OA in this process. NMR data indicated an equilibrium between free and bound OA, which shifts towards free OA as ELOA is progressively diluted, indicating that OA is relatively loosely bound. Activity measurements together with fluorescence spectroscopy and circular dichroism suggested a conversion of ELOA towards a more native-like state on interaction with lipid membranes, although complete refolding was not observed. Altogether, these results suggest that ELOA may act as an OA carrier and facilitate OA transfer to the membrane. ELOA's properties illustrate that protein folding variants may possess specific functional properties distinct from the native protein.     

Quantification of Key Genes Steering the Microbial Nitrogen Cycle in the Rhizosphere of Sorghum Cultivars in Tropical Agroecosystems

The effect of agricultural management practices on geochemical cycles in moderate ecosystems is by far better understood than in semiarid regions, where fertilizer availability and climatic conditions are less favorable. We studied the impact of different fertilizer regimens in an agricultural long-term observatory in Burkina Faso at three different plant development stages (early leaf development, flowering, and senescence) of sorghum cultivars. Using real-time PCR, we investigated functional microbial communities involved in key processes of the nitrogen cycle (nitrogen fixation, ammonia oxidation, and denitrification) in the rhizosphere. The results indicate that fertilizer treatments and plant development stages combined with environmental factors affected the abundance of the targeted functional genes in the rhizosphere. While nitrogen-fixing populations dominated the investigated communities when organic fertilizers (manure and straw) were applied, their numbers were comparatively reduced in urea-treated plots. In contrast, ammonia-oxidizing bacteria (AOB) increased not only in absolute numbers but also in relation to the other bacterial groups investigated in the urea-amended plots. Ammonia-oxidizing archaea exhibited higher numbers compared to AOB independent of fertilizer application. Similarly, denitrifiers were also more abundant in the urea-treated plots. Our data imply as well that, more than in moderate regions, water availability might shape microbial communities in the rhizosphere, since low gene abundance data were obtained for all tested genes at the flowering stage, when water availability was very limited.Land degradation is one of the most serious threats to food production on the African continent. Soil erosion, nutrient depletion, low organic matter content, and unfavorable pH values are some of the reasons for a deficient soil fertility (30), mainly in Central African countries. Combined with high variability and irregular distribution of rainfall, these factors contribute to negative nutrient balances. For example, 4.4 million tons of nitrogen (N) are lost per year in African soils, but only 0.8 million tons are reapplied by fertilization (12, 34). Since nitrogen is a key nutrient determining the productivity of agroecosystems (7, 11, 43), it is of central importance to optimize the nitrogen balance in these countries, mainly by steering the genetic resources of soil microbes in a way that losses of applied nitrogen are minimized and biological nitrogen fixation is increased. The aim should be to obtain a highly efficient nitrogen turnover, with leaching of nitrate and losses of gaseous products such as nitrous oxide (N₂O) or dinitrogen (N₂) as low as possible.Despite the importance of this issue, not much data are available on microbial community structure and function related to the nitrogen cycle in agroecosystems of Central Africa, and scenarios from moderate climatic regions cannot simply be transferred to tropical agroecosystems. Furthermore, the few studies published thus far only investigated effects of agricultural management on a single process of the nitrogen turnover (18, 20, 32) and ignore the fact that nitrogen turnover is a network of closely interlinked processes.Therefore, we sought to investigate the effects of different fertilizer regimens on multiple transformation processes within the nitrogen cycle in agroecosystems from semiarid areas in Central Africa. We investigated nitrogen dynamics on a full-cycle approach, including the most important steps in well-aerated agricultural soils (nitrification, denitrification, and nitrogen fixation). We hypothesized that each fertilizer regimen results in typical abundance pattern of the functional populations. While in moderate agroecosystems the plant (respectively, the plant development stage and the plant performance) plays an important role in shaping microbial community structure and function in the rhizosphere, we further postulated that nitrogen turnover and the corresponding populations are also influenced by the availability of water in semiarid soils. To test these hypotheses on a molecular basis, we quantified bacterial genes encoding the nitrogenase reductase (nifH), ammonia monooxygenase (amoA), and nitrite reductase (nirK and nirS), as well as archaeal amoA genes, by real-time PCR and linked the data to soil properties and grain yield.     

Dynamics and functional relevance of ammonia-oxidizing archaea in two agricultural soils

Crucial steps in geochemical cycles are in many cases performed by more than one group of microorganisms, but the significance of this functional redundancy with respect to ecosystem functioning is poorly understood. Ammonia-oxidizing archaea (AOA) and their bacterial counterparts (AOB) are a perfect system to address this question: although performing the same transformation step, they belong to well-separated phylogenetic groups. Using pig manure amended with different concentrations of sulfadiazine (SDZ), an antibiotic that is frequently used in veterinary medicine, it was possible to affect AOB and AOA to different degrees. Addition of manure stimulated growth of AOB in both soils and, interestingly, also growth of AOA was considerably stimulated in one of the soils. The antibiotic treatments decreased the manure effect notably on AOB, whereas AOA were affected to a lower extent. Model calculations concerning the respective proportions of AOA and AOB in ammonia oxidation indicate a substantial contribution of AOA in one of the soils that further increased under the influence of SDZ, hence indicating functional redundancy between AOA and AOB.