Soil net nitrogen mineralisation across global grasslands.

Soil nitrogen mineralisation (Nmin), the conversion of organic into inorganic N, is important for productivity and nutrient cycling. The balance between mineralisation and immobilisation (net Nmin) varies with soil properties and climate. However, because most global-scale assessments of net Nmin are laboratory-based, its regulation under field-conditions and implications for real-world soil functioning remain uncertain. Here, we explore the drivers of realised (field) and potential (laboratory) soil net Nmin across 30 grasslands worldwide. We find that realised Nmin is largely explained by temperature of the wettest quarter, microbial biomass, clay content and bulk density. Potential Nmin only weakly correlates with realised Nmin, but contributes to explain realised net Nmin when combined with soil and climatic variables. We provide novel insights of global realised soil net Nmin and show that potential soil net Nmin data available in the literature could be parameterised with soil and climate data to better predict realised Nmin.

Sensitivity of global soil carbon stocks to combined nutrient enrichment.

Soil stores approximately twice as much carbon as the atmosphere and fluctuations in the size of the soil carbon pool directly influence climate conditions. We used the Nutrient Network global change experiment to examine how anthropogenic nutrient enrichment might influence grassland soil carbon storage at a global scale. In isolation, enrichment of nitrogen and phosphorous had minimal impacts on soil carbon storage. However, when these nutrients were added in combination with potassium and micronutrients, soil carbon stocks changed considerably, with an average increase of 0.04 KgCm-2  year-1 (standard deviation 0.18 KgCm-2  year-1 ). These effects did not correlate with changes in primary productivity, suggesting that soil carbon decomposition may have been restricted. Although nutrient enrichment caused soil carbon gains most dry, sandy regions, considerable absolute losses of soil carbon may occur in high-latitude regions that store the majority of the world's soil carbon. These mechanistic insights into the sensitivity of grassland carbon stocks to nutrient enrichment can facilitate biochemical modelling efforts to project carbon cycling under future climate scenarios.

Androgenetic/biparental mosaicism causes placental mesenchymal dysplasia.

BACKGROUND: Placental mesenchymal dysplasia (PMD) is a distinct syndrome of unknown aetiology that is associated with significant fetal morbidity and mortality. Intrauterine growth restriction is common, yet, paradoxically, many of the associated fetuses/newborns have been diagnosed with Beckwith-Wiedemann syndrome (BWS). METHODS: We report two cases of PMD with high levels of androgenetic (complete paternal uniparental isodisomy) cells in the placenta and document, in one case, a likely androgenetic contribution to the fetus as well. RESULTS: The same haploid paternal complement found in the androgenetic cells was present in coexisting biparental cells, suggesting origin from a single fertilisation event. CONCLUSIONS: Preferential allocation of the normal cells into the trophoblast explains the absence of trophoblast overgrowth, a key feature of this syndrome. Interestingly, the distribution of androgenetic cells appears to differ from that reported for artificially created androgenetic mouse chimeras. Androgenetic mosaicism for the first time provides an aetiology for PMD, and may be a novel mechanism for BWS and unexplained intrauterine growth restriction.

Variation in growth rate and ecophysiology among 34 grassland and savanna species under contrasting N supply: a test of functional group differences.

• We tested the hypothesis that biological trait-based plant functional groups provide sufficient differentiation of species to enable generalization about a variety of plant ecophysiological traits or responses to nitrogen (N). • Seedlings of 34 North American grassland and savanna species, representing 5 functional groups, were grown in a glasshouse in an infertile soil with or without N fertilization. • Forbs, C3 and C4 grasses, on average, had similar relative growth rates (RGR), followed in declining order by legumes and oaks, but RGR varied greatly among species within functional groups. All measured attributes differed significantly among functional groups, of these, only RGR and photosynthesis differed among functional groups in response to N. All groups, except the legumes, had significantly greater photosynthetic and respiration rates at elevated N supply. Principal components analyses and cluster analyses yielded groupings that corresponded only moderately well to the biologically based a priori functional groupings. • Variation in RGR among species and treatments was positively related to net CO2 exchange (photosynthesis and respiration) and net assimilation rate, but unrelated to leaf area ratio. Photosynthetic and respiration rates were related to tissue %N among treatments and species. Our data indicate that RGR and related traits differ among the functional groups in significant ways, but in a complex pattern that does not yield simple generalizations about relative performance, controls on RGR, or response to resource supply rate.

Diversity and productivity in a long-term grassland experiment.

Plant diversity and niche complementarity had progressively stronger effects on ecosystem functioning during a 7-year experiment, with 16-species plots attaining 2.7 times greater biomass than monocultures. Diversity effects were neither transients nor explained solely by a few productive or unviable species. Rather, many higher-diversity plots outperformed the best monoculture. These results help resolve debate over biodiversity and ecosystem functioning, show effects at higher than expected diversity levels, and demonstrate, for these ecosystems, that even the best-chosen monocultures cannot achieve greater productivity or carbon stores than higher-diversity sites.

Plant diversity enhances ecosystem responses to elevated CO2 and nitrogen deposition.

Human actions are causing declines in plant biodiversity, increases in atmospheric CO2 concentrations and increases in nitrogen deposition; however, the interactive effects of these factors on ecosystem processes are unknown. Reduced biodiversity has raised numerous concerns, including the possibility that ecosystem functioning may be affected negatively, which might be particularly important in the face of other global changes. Here we present results of a grassland field experiment in Minnesota, USA, that tests the hypothesis that plant diversity and composition influence the enhancement of biomass and carbon acquisition in ecosystems subjected to elevated atmospheric CO2 concentrations and nitrogen deposition. The study experimentally controlled plant diversity (1, 4, 9 or 16 species), soil nitrogen (unamended versus deposition of 4 g of nitrogen per m2 per yr) and atmospheric CO2 concentrations using free-air CO2 enrichment (ambient, 368 micromol mol-1, versus elevated, 560 micromol mol-1). We found that the enhanced biomass accumulation in response to elevated levels of CO2 or nitrogen, or their combination, is less in species-poor than in species-rich assemblages.

Intra- and intermolecular triplex DNA formation in the murine c-myb proto-oncogene promoter are inhibited by mithramycin.

Mithramycin inhibits transcription by binding to G/C-rich sequences, thereby preventing regulatory protein binding. However, it is also possible that mithramycin inhibits gene expression by preventing intramolecular triplex DNA assembly. We tested this hypothesis using the DNA triplex adopted by the murine c-myb proto-oncogene. The 5'-regulatory region of c-myb contains two polypurine:polypyrimidine tracts with imperfect mirror symmetry, which are highly conserved in the murine and human c-myb sequences. The DNA binding drugs mithramycin and distamycin bind to one of these regions as determined by DNase I protection assay. Gel mobility shift assays, nuclease and chemical hypersensitivity and 2D-gel topological analyses as well as triplex-specific antibody binding studies confirmed the formation of purine*purine:pyrimidine inter- and pyrimidine*purine:pyrimidine intra-molecular triplex structures in this sequence. Mithramycin binding within the triplex target site displaces the major groove-bound oligonucleotide, and also abrogates the supercoil-dependent H-DNA formation, whereas distamycin binding had no such effects. Molecular modeling studies further support these observations. Triplex-specific antibody staining of cells pretreated with mithramycin demonstrates a reversal of chromosomal triplex structures compared to the non-treated and distamycin-treated cells. These observations suggest that DNA minor groove-binding drugs interfere with gene expression by precluding intramolecular triplex formation, as well as by physically preventing regulatory protein binding.

Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain.

Converging lines of evidence implicate the beta-amyloid peptide (Ass) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce A beta production by functionally inhibiting gamma-secretase, the activity responsible for the carboxy-terminal cleavage required for A beta production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon A beta production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, to mice transgenic for human APP(V717F) reduces brain levels of Ass in a dose-dependent manner within 3 h. These studies represent the first demonstration of a reduction of brain A beta in vivo. Development of such novel functional gamma-secretase inhibitors will enable a clinical examination of the A beta hypothesis that Ass peptide drives the neuropathology observed in Alzheimer's disease.

Contrasting effects of plant richness and composition on insect communities: a field experiment.

We experimentally separated the effects of two components of plant diversity-plant species richness and plant functional group richness-on insect communities. Plant species richness and plant functional group richness had contrasting effects on insect abundances, a result we attributed to three factors. First, lower insect abundances at higher plant functional group richness were explained by a sampling effect, which was caused by the increasing likelihood that one low-quality group, C4 grasses, would be present and reduce average insect abundances by 25%. Second, plant biomass, which was positively related to plant functional group richness, had a strong, positive effect on insect abundances. Third, a positive effect of plant species richness on insect abundances may have been caused by greater availability of alternate plant resources or greater vegetational structure. In addition, a greater diversity of insect species, whose individual abundances were often unaffected by changes in plant species richness, may have generated higher total community abundances. After controlling for the strong, positive influence of insect abundance on insect diversity through rarefaction, insect species richness increased as plant species richness and plant functional group richness increased. Although these variables did not explain a high proportion of variation individually, plant species richness and plant functional group richness had similar effects on insect diversity and opposing effects on insect abundances, and both factors may explain how the loss of plant diversity influences higher trophic levels.

In situ analysis of the transcriptional activity of integrated viral DNA using tyramide-FISH.

Infection by the oncogenic human papillomavirus (HPV) types 16 and type 18 can progress to cancers. Two well studied cervical carcinoma cell lines, SiHa and CaSki, contain two to four copies, or several hundred copies of integrated HPV-16, respectively. To define the chromosomal loci from which HPV mRNAs are transcribed in these cells, we have simultaneously visualized chromosomal DNA territories, HPV DNA or nascent HPV RNA sequences by using a highly sensitive in situ hybridization (T-FISH) technique employing deposition of fluorescent tyramides. We found that, in SiHa cells, nascent HPV RNAs co-localized with both integrated HPV copies on chromosome 13. Surprisingly, in CaSki cells, nascent HPV RNA only co-localized with one minor HPV DNA-positive locus on chromosome 14. The DNA signal intensity of this locus was consistent with a single to a few HPV intergrants. The tyramide methodologies described here provide an in-depth molecular cytological analyses applicable to research and diagnosis.

The value of CAGE, CUGE, and AUDIT in screening for alcohol abuse and dependence among college freshmen.

BACKGROUND: This study attempted to (1) determine the prevalence of alcohol problems in college freshmen, (2) assess the performance of both the CAGE and the Alcohol Use Disorders Identification Test (AUDIT) questionnaires in this population, and (3) assess the possibility of improving the CAGE and/or AUDIT. METHODS: A sample of 3564 consecutive college freshmen, with a mean age of 18 years, at the Catholic University of Leuven, (Belgium) completed, during a cross-sectional study, a questionnaire assessing drinking behavior and identifying students at risk as defined by DSM-IV criteria. The questionnaire also included the CAGE questionnaire and the AUDIT. Calculations of sensitivity, specificity, negative predictive value, positive predictive value, likelihood ratios, and receiver operating characteristic curves for different scores of the CAGE and the AUDIT were performed, using DSM-IV criteria as the reference standard. RESULTS: The area under the receiver operating characteristic curve of the CAGE and the AUDIT was 0.76 and 0.85, respectively. The cutoff score of 1 for the CAGE was associated with a sensitivity of 42%, a specificity of 87%, a positive predictive value of 36%, and a negative predictive value of 90%. A score of 6 or more for the AUDIT gave a sensitivity of 80%, a specificity of 78%, a positive predictive value of 37%, and a negative predictive value of 77%. These results were related with a prevalence of 14.1% of alcohol problems. Replacing one question of the CAGE by "often driving under the influence" resulted in the CUGE (acronym for "cut down, under influence, guilty feelings, and eye opener"), with an area under the curve of 0.96, a positive likelihood ratio of 8.7, and a negative likelihood ratio of 0.04. CONCLUSIONS: Prevalence of alcohol problems in college students is confirmed to be high. When screening for alcohol problems in a college freshmen population, one question seems extremely important. The newly constructed CUGE questionnaire may improve screening efforts in students, compared with existing questionnaires.

Purification and cloning of amyloid precursor protein beta-secretase from human brain.

Proteolytic processing of the amyloid precursor protein (APP) generates amyloid beta (Abeta) peptide, which is thought to be causal for the pathology and subsequent cognitive decline in Alzheimer's disease. Cleavage by beta-secretase at the amino terminus of the Abeta peptide sequence, between residues 671 and 672 of APP, leads to the generation and extracellular release of beta-cleaved soluble APP, and a corresponding cell-associated carboxy-terminal fragment. Cleavage of the C-terminal fragment by gamma-secretase(s) leads to the formation of Abeta. The pathogenic mutation K670M671-->N670L671 at the beta-secretase cleavage site in APP, which was discovered in a Swedish family with familial Alzheimer's disease, leads to increased beta-secretase cleavage of the mutant substrate. Here we describe a membrane-bound enzyme activity that cleaves full-length APP at the beta-secretase cleavage site, and find it to be the predominant beta-cleavage activity in human brain. We have purified this enzyme activity to homogeneity from human brain using a new substrate analogue inhibitor of the enzyme activity, and show that the purified enzyme has all the properties predicted for beta-secretase. Cloning and expression of the enzyme reveals that human brain beta-secretase is a new membrane-bound aspartic proteinase.

The effect of reconstitution of an Haemophilus influenzae type b-tentanus toxoid conjugate (PRP-T) vaccine on the immune responses to a diphtheria-tetanus-whole cell pertussis (DTwP) vaccine: a five-year follow-up.

Controversial results have been obtained from previous studies on the combined administration of Haemophilus influenzae type b-tetanus toxoid conjugate (PRP-T) and diphtheria-tetanus-whole-cell pertussis (DTwP) combination vaccines, with regard to possible reciprocal interference between the constituent antigens. To document the priming effect and possible long-term immunogenic interference of PRP-T and DTwP combination vaccines, a randomized, double-blind, controlled study was conducted in Belgium. A total of 168 healthy infants received, at 3, 4 and 5 months of age, DTwP vaccine mixed just prior to injection either with PRP-T vaccine (group A, DTwP//PRP-T, N = 85) or with placebo (group B, DTwP//Placebo, N = 83). At the age of 14 months, children of both groups were randomized to receive either a dose of DTwP//PRP-T vaccine (subgroups A1 and B1) or a dose of Hib polysaccharide (PRP) vaccine (subgroups A2 and B2). Those children in subgroups A1 and B1 had an additional serum sample taken at the age of 5 years (at the time of a DT booster). The immune response to Hib polysaccharide at the age of 4, 5 and 6 months confirmed the excellent immunogenicity profile of PRP-T in infants. In addition, the vigorous anamnestic response (i.e. a 20-fold increase of GMT) to a booster dose of the plain capsular polysaccharide (PRP) reflected the efficient Hib-priming induced by the combined DTwP//PRP-T vaccine. Reconstitution of PRP-T with DTwP did not affect the immune response to diphtheria toxoid or pertussis agglutinins. Nevertheless, at almost any time point during the five-year follow-up, the tetanus antitoxin GMT values were significantly lower in the DTwP//PRP-T group (A and A1) than in the DTwP//Placebo group (B and B1). Despite the suppressive effect on GMT values, intergroup differences in rates of seroprotection were never significant, except after doses 2 and 3 for which there were lower percentages of children in group A with antitoxin titers > 0.05 IU/mL and > 1.0 IU/mL. In the group primed with the combined DTwP//PRP-T vaccine, (1) a DT booster dose at the age of 5 years provoked a 150-fold increase in tetanus antitoxin GMT, (2) a high tetanus antitoxin GMT value was attained (GMT = 19.3 IU/mL) and (3) all children in this group had tetanus antitoxin titers > 1.0 IU/mL, so it may be concluded that all these children will still be protected against tetanus until at least the age of the next recommended booster dose (i.e. the age of 15 years). No differences in the occurrence of adverse events were observed between the groups who received the DTwP//PRP-T vaccine or the DTwP//Placebo vaccine, both vaccines being associated with events customarily attributable to DTwP (data not shown). Our results indicate (1) that the combination vaccine, DTwP//PRP-T, represents a safe and effective alternative for the existing uncombined vaccines and (2) that the long-term effect of interference between the components of future combination vaccines should be studied with subsequent booster doses, followed by the evaluation of persistence of antibodies over several years.

Effects of plant species richness on invasion dynamics, disease outbreaks, insect abundances and diversity.

Declining biodiversity represents one of the most dramatic and irreversible aspects of anthropogenic global change, yet the ecological implications of this change are poorly understood. Recent studies have shown that biodiversity loss of basal species, such as autotrophs or plants, affects fundamental ecosystem processes such as nutrient dynamics and autotrophic production. Ecological theory predicts that changes induced by the loss of biodiversity at the base of an ecosystem should impact the entire system. Here we show that experimental reductions in grassland plant richness increase ecosystem vulnerability to invasions by plant species, enhance the spread of plant fungal diseases, and alter the richness and structure of insect communities. These results suggest that the loss of basal species may have profound effects on the integrity and functioning of ecosystems.

Localization of HuC (ELAVL3) to chromosome 19p13.2 by fluorescence in situ hybridization utilizing a novel tyramide labeling technique.

HuC is a neural-specific member of the Elav family of RNA-binding proteins. This highly conserved gene family plays a crucial role in neurogenesis, and HuC (HGMW-approved symbol ELAVL3) is expressed at an early stage of neural development. Using a novel tyramide fluorescence in situ hybridization (T-FISH) technique, we localized HuC to chromosome 19p13.2. This localization was confirmed by radiation hybrid mapping and coincides with that of HuR (HGMW-approved symbol ELAVL1), another elav family member. Dual T-FISH analysis with HuC and HuR probes, however, indicated distinct loci, with HuC being centromeric to HuR. This study demonstrates the utility of T-FISH in colocalizing two genes on the same chromosomal preparation using only biotinylated probes.

Bronchodilator response in 3-6.5 years old healthy and stable asthmatic children.

Few data are available on the bronchodilator response in preschool children. This study was set up to study baseline lung function and bronchodilator responses in healthy and asthmatic children younger than 7 yrs old. In 281 preschool children attending kindergarten (age range 2.7-6.6 yrs old) respiratory system resistance (Rrs) and reactance (Xrs) by impulse oscillation system at 5, 10, 15, 20, 25 and 35 Hz as well as resonance frequency (f0) were measured before and 20 min after 200 microg inhaled salbutamol by a metered-dose inhaler connected to a spacer device. Thirty-four of them were diagnosed as asthmatics based on a validated standardized questionnaire. The mean Rrs (+/-SD) at 5 Hz (Rrs,5) was 1.03 (+/-0.24) kPa x L(-1) x s for healthy children and 1.09 (+/-0.26) kPa x L(-1) x s for stable asthmatics. The mean change in Rrs,5 after salbutamol was -0.13 (+/-0.20) kPa x L(-1) x s for the healthy children and -0.09 (+/-0.25) kPa x L(-1) x s for the asthmatic group. The scatter for the measurements was striking. Neither baseline values of impulse oscillation nor its changes after bronchodilator administration was significantly different between healthy and stable asthmatic children. A change in respiratory system resistance at 5 Hz of 40% is to be considered the cut-off for a " positive" bronchodilator response.

Safety and immunogenicity of an Haemophilus influenzae type b-tetanus toxoid conjugate (PRP-T) and diphtheria-tetanus-pertussis (DTP) combination vaccine administered in a dual-chamber syringe to infants in Belgium and Chile.

To document any unexpected differences in the immune response between study populations and to evaluate immunogenicity and safety of a simplified presentation (dual-chamber syringe) of an Haemophilus influenzae type b-tetanus toxoid conjugate (PRP-T) and diphtheria-tetanus-pertussis (DTP) combination vaccine, a multicentre, randomized, comparative study was conducted in Belgium and Chile. A total of 537 healthy infants, 270 in Chile and 267 in Belgium, received PRP-T and DTP vaccines combined in a dual-chamber syringe (D-Ch group, DTP/PRP-T, reconstituted by pressing the plunger of the syringe immediately before injection, n = 239) or combined in a single-chamber syringe (C-In group, DTP@PRP-T, reconstituted immediately before injection, n = 61) or in separate injections (S-In group, DTP + PRP-T, simultaneously injected at separate sites, n = 237) at 3, 4, and 5 months of age. Serum samples were collected before vaccination and at 6 months of age. In the D-Ch group, the incidence of adverse events was comparable to administration of DTP vaccine alone. Higher rates of local and systemic reactions were observed in the Chilean population, possibly due to differences in surveillance practice. The immune response to each vaccine component compared well to that of the separate administration of PRP-T and DTP vaccines, except for higher post-immunization anti-PRP geometric mean titre (GMT) values after separate injections (25.6 micrograms mL-1) than after combined injection with the dual-chamber syringe (17.6 micrograms mL-1) (p = 0.001). An unexpected 'syringe' effect was seen: a greater post-immunization anti-PRP GMT was observed in the D-Ch group (17.6 micrograms mL-1) than in the C-In group (7.7 micrograms mL-1) (p = 0.0001). Whereas pre-immunization GMTs of some antibodies were significantly lower in Chilean than in Belgian infants, the post-immunization GMTs of Chilean infants were two to three times greater for all of the antibodies studied (p < 0.005). Differences in reactogenicity and in the immune response between the study populations or the different vaccine presentations were striking, but are probably of no clinical relevance. The convenient dual-chamber syringe presentation of DTP and PRP-T vaccines is safe and highly immunogenic.