Nitrogen Cycling and Mass Balance for a Forested Catchment in the Italian Alps. Assessment of Nitrogen Status

During 1999–2001 the chemical composition and fluxes were measured in rainfall, throughfall, soil solution and stream water in a remote forested site in the Italian Alps. The analysis of temporal patterns revealed the differential behaviour of nitrogen and sulphur and suggested that different mechanisms controlled their flux. No important changes in sulphate concentration and fluxes emerged as the solution passed through the various components of the forest ecosystem, and temporal variations of SO₄ in the soil solution and stream were likely driven by the physical process of dilution. The availability of nitrate and ammonia, by contrast, was drastically reduced as throughfall water entered the soil and passed through the mineral layers, irrespective of season. The calculated hydrochemical budget based on throughfall and soil solution N fluxes revealed that ~80% N retention in the forest soil, corresponding to 12 kg ha⁻¹ yr⁻¹, despite a relatively high N deposition loading (15 kg ha⁻¹ yr⁻¹). Most of the leached nitrogen (90%) was in the organic form. Indicators of the N status of this ecosystem, such as C/N ratio in solid and solution phase of the soil and N foliage content as well as land use history were examined. Despite the strong N retention in the forested part of the catchment, the stream water N–NO₃ levels were consistently above 10 μg l⁻¹ suggesting that the Val Masino catchment as a whole was less efficient in processing atmospheric N inputs. This contrasting N behaviour illustrates the role of landscape features, such as the soil cover and vegetation type, that is characteristic of an alpine catchment.     

In vitro effects of GSM modulated radiofrequency fields on human immune cells

Despite the important role of the immune system in defending the body against infections and cancer, only few investigations on possible effects of radiofrequency (RF) radiation on function of human immune cells have been undertaken. Aim of the present investigation was therefore to assess whether GSM modulated RF fields have adverse effects on the functional competence of human immune cells. Within the frame of the multidisciplinary project "Biological effects of high frequency electromagnetic fields (EMF)" sponsored by the National Occupation Hazard Insurance Association (AUVA) in vitro investigations were carried out on human blood cells. Exposure was performed at GSM Basic 1950 MHz, an SAR of 1 mW/g in an intermittent mode (5 min "ON", 10 min "OFF") and a maximum Delta T of 0.06 degrees C for the duration of 8 h. The following immune parameters were evaluated: (1) the intracellular production of interleukin-2 (IL-2) and interferon (INF) gamma in lymphocytes, and IL-1 and tumor necrosis factor (TNF)-alpha in monocytes were evaluated with monoclonal antibodies. (2) The activity of immune-relevant genes (IL 1-alpha and beta, IL-2, IL-2-receptor, IL-4, macrophage colony stimulating factor (MCSF)-receptor, TNF-alpha, TNF-alpha-receptor) and housekeeping genes was analyzed with real time PCR. (3) The cytotoxicity of lymphokine activated killer cells (LAK cells) against a tumor cell line was determined in a flow cytometric test. For each parameter, blood samples of at least 15 donors were evaluated. No statistically significant effects of exposure were found and there is no indication that emissions from mobile phones are associated with adverse effects on the human immune system.     

Genetic analysis of candidate genes modifying the age-at-onset in Huntington’s disease

The expansion of a polymorphic CAG repeat in the HD gene encoding huntingtin has been identified as the major cause of Huntington’s disease (HD) and determines 42–73% of the variance in the age-at-onset of the disease. Polymorphisms in huntingtin interacting or associated genes are thought to modify the course of the disease. To identify genetic modifiers influencing the age at disease onset, we searched for polymorphic markers in the GRIK2, TBP, BDNF, HIP1 and ZDHHC17 genes and analysed seven of them by association studies in 980 independent European HD patients. Screening for unknown sequence variations we found besides several silent variations three polymorphisms in the ZDHHC17 gene. These and polymorphisms in the GRIK2, TBP and BDNF genes were analysed with respect to their association with the HD age-at-onset. Although some of the factors have been defined as genetic modifier factors in previous studies, none of the genes encoding GRIK2, TBP, BDNF and ZDHHC17 could be identified as a genetic modifier for HD.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Time-dependent effects of sodium arsenite on DNA breakage and apoptosis observed in the comet assay

To assess genotoxic effects of sodium arsenite (NaAsO2) the single-cell gel electrophoresis (comet assay) had been conducted in various studies indicating genotoxicity. However, DNA fragmentation due to NaAsO2-induced apoptosis may constitute a bias in the interpretation of the results. Apoptotic cells can show typically large and diffuse comets, which are usually excluded during genotoxicity analysis. It is controversial whether there is a time-window in which the apoptotic process generates comets that would falsely be interpreted to be the result of genotoxic DNA damage. Therefore, we evaluated frequency histograms for single-cell measures of tail DNA (% DNA in comet tail) in 30-min intervals after incubation of mouse lymphoma L5178Y cells with sodium arsenite (NaAsO2). In parallel, we evaluated apoptosis by measuring annexin V-positive cells with flow cytometry, and visualized apoptotic cells on slides by Hoechst bisbenzimide 33258 staining. The first observed effect at 30 min after treatment was an increase in annexin V-positive cells. At about 60 min the number of cells with moderate DNA migration increased in the comet-assay analysis. After 90 min, an increase in the number of cells with high levels of DNA migration was observed, which resulted in a bimodal distribution of cells with moderate and high levels of DNA migration. Hoechst-stained apoptotic cells could only be observed at later times (> or = 120 min). This means that the treatment would have been considered to be genotoxic if analysed at 120 min even if the cells with high levels of DNA migration would have been excluded. The occurrence of annexin V-positive cells preceded the appearance of cells with moderate levels of DNA migration. We hypothesize that these cells were early apoptotic cells and not indicative of genotoxic damage. We conclude that DNA-damaging effects of NaAsO2 cannot adequately be interpreted if the comet assay is not accompanied by separate analysis of early endpoints for induction of apoptosis.     

Cannabidiol inhibits lung cancer cell invasion and metastasis via intercellular adhesion molecule-1

Cannabinoids inhibit cancer cell invasion via increasing tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). This study investigates the role of intercellular adhesion molecule-1 (ICAM-1) within this action. In the lung cancer cell lines A549, H358, and H460, cannabidiol (CBD; 0.001-3 μM) elicited concentration-dependent ICAM-1 up-regulation compared to vehicle via cannabinoid receptors, transient receptor potential vanilloid 1, and p42/44 mitogen-activated protein kinase. Up-regulation of ICAM-1 mRNA by CBD in A549 was 4-fold at 3 μM, with significant effects already evident at 0.01 μM. ICAM-1 induction became significant after 2 h, whereas significant TIMP-1 mRNA increases were observed only after 48 h. Inhibition of ICAM-1 by antibody or siRNA approaches reversed the anti-invasive and TIMP-1-upregulating action of CBD and the likewise ICAM-1-inducing cannabinoids Δ(9)-tetrahydrocannabinol and R(+)-methanandamide when compared to isotype or nonsilencing siRNA controls. ICAM-1-dependent anti-invasive cannabinoid effects were confirmed in primary tumor cells from a lung cancer patient. In athymic nude mice, CBD elicited a 2.6- and 3.0-fold increase of ICAM-1 and TIMP-1 protein in A549 xenografts, as compared to vehicle-treated animals, and an antimetastatic effect that was fully reversed by a neutralizing antibody against ICAM-1 [% metastatic lung nodules vs. isotype control (100%): 47.7% for CBD + isotype antibody and 106.6% for CBD + ICAM-1 antibody]. Overall, our data indicate that cannabinoids induce ICAM-1, thereby conferring TIMP-1 induction and subsequent decreased cancer cell invasiveness.     

Dietary intake of plant sterols stably increases plant sterol levels in the murine brain

Plant sterols such as sitosterol and campesterol are frequently administered as cholesterol-lowering supplements in food. Recently, it has been shown in mice that, in contrast to the structurally related cholesterol, circulating plant sterols can enter the brain. We questioned whether the accumulation of plant sterols in murine brain is reversible. After being fed a plant sterol ester-enriched diet for 6 weeks, C57BL/6NCrl mice displayed significantly increased concentrations of plant sterols in serum, liver, and brain by 2- to 3-fold. Blocking intestinal sterol uptake for the next 6 months while feeding the mice with a plant stanol ester-enriched diet resulted in strongly decreased plant sterol levels in serum and liver, without affecting brain plant sterol levels. Relative to plasma concentrations, brain levels of campesterol were higher than sitosterol, suggesting that campesterol traverses the blood-brain barrier more efficiently. In vitro experiments with brain endothelial cell cultures showed that campesterol crossed the blood-brain barrier more efficiently than sitosterol. We conclude that, over a 6-month period, plant sterol accumulation in murine brain is virtually irreversible.     

Haptoglobin baseline value in jennies and the effect of ovariectomy on its serum concentration

The study was conducted to determine the baseline concentration of serum haptoglobin (Hp) in jennies during the breeding and nonbreeding season and to evaluate the effects of ovariectomy on serum Hp concentrations in jennies. Eighteen adult jennies were divided in three groups: nine jennies (OVA) were ovariectomized using laparoscopic surgery, six jennies (LAP) were exploratory examined by laparoscopic surgery, and three jennies were used as a control group. Blood samples were collected from the animals at Day -6, -2, -1, 0, 1, 2, 5, 8, 15, 22, 29 and 36 of surgery. Serum samples were analyzed by an ELISA specifically developed for determining equine Hp. The mean weekly Hp concentration ranged between 149.76 ± 7.55 and 178.94 ± 6.67 mg/L. The Hp concentrations of clinically healthy jennies revealed no significant variation among time, and there was no effect of reproductive season on Hp concentrations in jennies. Serum Hp concentration was elevated at the first day after operations in the OVA and LAP group. Five days after the operation, the Hp concentration reached the maximum in the LAP and OVA group (278.84 ± 34.22 and 359.88 ± 35.45 mg/L, respectively) and decreased at Day 8 after the operations. On Day 22, 29 and 32 after the operations, the concentration of Hp in LAP and OVA animals was close to its concentration in the control group. In conclusion, Hp is not related to reproductive status of jennies and it can be used as an indicator for cell and tissue damage after surgical operations.     

Role of tartrate-resistant acid phosphatase (TRAP) in long bone development

Tartrate resistant acid phosphatase (TRAP) was shown to be critical for skeleton development, and TRAP deficiency leads to a reduced resorptive activity during endochondral ossification resulting in an osteopetrotic phenotype and shortened long bones in adult mice. A proper longitudinal growth depends on a timely, well-coordinated vascularization and formation of the secondary ossification center (SOC) of the long bones epiphysis. Our results demonstrate that TRAP is not essential for the formation of the epiphyseal vascular network. Therefore, in wild type (Wt) controls as well as TRAP deficient (TRAP(-/-)) mutants vascularised cartilage canals are present from postnatal day (P) five. However, in the epiphysis of the TRAP(-/-) mice cartilage mineralization, formation of the marrow cavity and the SOC occur prematurely compared with the controls. In the mutant mice the entire growth plate is widened due to an expansion of the hypertrophic zone. This is not seen in younger animals but first detected at week (W) three and during further development. Moreover, an enhanced number of thickened trabeculae, indicative of the osteopetrotic phenotype, are observed in the metaphysis beginning with W three. Epiphyseal excavation was proposed as an important function of TRAP, and we examined whether TRAP deficiency affects this process. We therefore evaluated the marrow cavity volume (MCV) and the epiphyseal volume (EV) and computed the MCV to EV ratio (MCV/EV). We investigated developmental stages until W 12. Our results indicate that both epiphyseal excavation and establishment of the SOC are hardly impaired in the knockouts. Furthermore, no differences in the morphology of the epiphyseal bone trabeculae and remodeling of the articular cartilage layers are noted between Wt and TRAP(-/-) mice. We conclude that in long bones, TRAP is critical for the development of the growth plate and the metaphysis but apparently not for the epiphyseal vascularization, excavation, and establishment of the SOC.