A broad-scale spatial analysis of the environmental benefits of fertiliser closed periods implemented under the Nitrates Directive in Europe.

Nutrient pollution from agriculture has been an ongoing challenge for decades, contributing to numerous negative environmental impacts. In the European Union policies have been developed to address nutrient pollution, including Nitrate Action Programmes under Council Directive 91/676/EEC. Although Member States report on progress on implementation, there have been few studies that explore how measures have been implemented; the environmental implications of any differences; and how they vary spatially on a European scale. This study aims to address this gap with respect to fertiliser closed periods (1155 different closed periods across 69 Nitrate Action Programmes). This included the development of an approach that can be applied using readily available spatial data. Each closed period was scored for its coverage of risk periods for losses of nitrate; organic material; nitrous oxide and ammonia. Closed periods were then matched to relevant combinations of spatial data for each environmental zone and fertiliser type. The scores for each combination were used to create maps and calculate spatial statistics. The results show that in addition to nitrate, closed periods also reduce the risk of organic material run-off, emissions of nitrous oxide and to a lesser extent ammonia. However, risk reduction is spatially variable across all the impacts and the scope for synergy is also variable (e.g. nitrate loss does not always correlate with nitrous oxide or ammonia risk reduction). Regions in the Atlantic, Lustanian and some areas within the Mediterranean zones appear to provide the greatest combined risk reduction, with other zones, especially in eastern Europe, having a lower combined risk reduction (due to a combination of different risk periods coupled with lower coverage of individual risks). The spatial analysis within this study is relatively simple; is based on a snapshot of closed periods during 2019-2020; and only explores one measure. However, it does provide some useful data and insights that could support policy development in the future. This includes scope for Member States and regions to learn from others where greater coverage of risk periods has been achieved; and highlighting how a more holistic perspective can be taken to the environmental management of nutrients. As we strive towards developing sustainable production systems, farmers and policy makers need to take a more integrated approach to incorporate additional environmental objectives; which increases the complexity of the challenge. Consequently, the demand for pragmatic approaches that take a more holistic approach is likely to increase in the future.

The iron status at birth of neonates with risk factors for developing iron deficiency: a pilot study.

OBJECTIVE: Small-for-gestational-age (SGA) neonates, infants of diabetic mothers (IDM) and very-low-birth weight premature neonates (VLBW) are reported to have increased risk for developing iron deficiency and possibly associated neurocognitive delays. STUDY DESIGN: We conducted a pilot study to assess iron status at birth in at-risk neonates by measuring iron parameters in umbilical cord blood from SGA, IDM, VLBW and comparison neonates. RESULTS: Six of the 50 infants studied had biochemical evidence of iron deficiency at birth. Laboratory findings consistent with iron deficiency were found in one SGA, one IDM, three VLBW, and one comparison infant. None of the infants had evidence of iron deficiency anemia. CONCLUSIONS: Evidence of biochemical iron deficiency at birth was found in 17% of screened neonates. Studies are needed to determine whether these infants are at risk for developing iron-limited erythropoiesis, iron deficiency anemia or iron-deficient neurocognitive delay.

Endogenous production and elevated levels of long-chain n-3 fatty acids in the milk of transgenic mice.

N-3 Polyunsaturated fatty acids (n-3 PUFA) are important for the normal development and functioning of all organisms. Mammals lack the n-3 fatty acid desaturase required for the synthesis of alpha-linolenic acid (18:3n-3), and are therefore dependent on dietary sources to obtain this essential fatty acid. Currently, the richest source of dietary long-chain n-3 PUFA, eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3), are triacylglycerides extracted from rapidly declining marine resources. The nematode Caenorhabditis elegans synthesizes a wide range of PUFA and possesses the only known example of an n-3 fatty acid desaturase enzyme in the animal kingdom. Transgenic mice expressing the C. elegans n-3 desaturase under the control of the lactation-induced goat beta-casein mammary gland promoter were generated via pronuclear microinjection. Significant increases in n-3 PUFA, decreases in n-6 PUFA, and an overall decrease in the n-6:n-3 PUFA ratio were observed in the milk produced by transgenic mice. Neonate mice consuming milk from transgenic females accumulated increased levels of docosahexaenoic acid in their brains. This transgenic model may provide useful information to address some basic questions of neonatal nutrition, and demonstrates one of the steps that would be required to increase the n-3 PUFA content of milk and dairy products endogenously. Increasing the proportion of n-3 PUFA in milk fat would help to improve the nutritional composition of an important component of the North American diet.

Phospholipase A2 domain formation in hydrolyzed asymmetric phospholipid monolayers at the air/water interface.

Phospholipase A2 (PLA2) catalyzed hydrolysis of asymmetric 1-caproyl-2-palmitoyl-phosphatidylcholine (6,16-PC) and 1-palmitoyl-2-caproyl-phosphatidylcholine (16,6-PC) lipid monolayers at the air/water interface was investigated. Surface pressure isotherms, surface potential and fluorescence microscopy at the air/water interface were used to characterize the asymmetric monolayer systems. Cobra (N. naja naja) and bee venom PLA2 exhibit hydrolytic activity towards 16,6-PC monolayers at all surface pressures up to monolayer collapse (37 mN m-1). Pancreatic PLA2 hydrolytic activity, however, was observed to be blocked at a lateral surface pressure of approx. 18 mN m-1 for both 6,16-PC and 16,6-PC monolayers. For 6,16-PC monolayers, fluorescence microscopy revealed that monolayer hydrolysis by PLA2 from cobra, bee, and bovine pancreatic sources all produced monolayer microstructuring. Fluorescence microscopy also showed that PLA2 is bound to these monolayer microstructures. Very little PLA2-induced microstructuring was observed to occur in 16,6-PC monolayer systems where caproic acid (C6) hydrolysis products were readily solubilized in the aqueous monolayer subphase. Surface potential measurements for 16,6-PC monolayer hydrolysis indicate dissolution of caproic acid reaction products into the monolayer subphase. Monolayer molecular area as a function of 6,16-PC monolayer hydrolysis time indicates the presence of monolayer-resident palmitic acid reaction products. With bovine serum albumin present in the monolayer subphase, PLA2 domain formation was observed only in hydrolyzed 6,16-PC monolayers. These results are consistent with laterally phase separated monolayer regions containing phospholipid and insoluble fatty acid reaction products from PLA2 monolayer hydrolysis electrostatically driving PLA2 adsorption to and enzyme domain formation at the heterogeneous, hydrolyzed lipid monolayer interface.

Characterization of gonadal sex cord-stromal tumor cell lines from inhibin-alpha and p53-deficient mice: the role of activin as an autocrine growth factor.

Inhibin-alpha-deficient mutant mice have been generated by a targeted deletion of the inhibin-alpha gene through homologous recombination in murine embryonic stem cells. Essentially all of the homozygous mutants develop gonadal sex cord-stromal tumors. To investigate their endocrine and proliferative characteristics, gonadal tumor cells were maintained in vitro. Cells from inhibin-alpha-deficient mice multiplied poorly; however, cells from mice deficient in both inhibin-alpha and p53 proliferated rapidly and showed higher saturation density and plating efficiency, thus allowing the establishment of clonal tumor cell lines. Although negligible estrogen and testosterone was produced by the clonal cells, high levels of progesterone were secreted. A clonal testis tumor cell line (inhibin-alpha/p53 deficient) showed no response to exogenous FSH, human CG (hCG), or inhibin A but exhibited a 6- to 8-fold increase in progesterone production in response to forskolin treatment. The stimulatory effect of forskolin was, however, partially blocked by activin treatment. Northern blot analysis revealed inhibin beta A and beta B mRNA expression in these cells. Furthermore, Western blot analyses indicated the secretion of the beta A-subunit protein. We further tested the role of activin on tumor cell growth. Treatment with follistatin, an activin-binding protein, inhibited tumor cell replication in a dose-dependent manner. In contrast, treatment with activin A stimulated tumor cell growth by itself and partially blocked follistatin action. Incorporation of thymidine into DNA of these cells was also stimulated by activin. In addition, treatment with antiactivin A serum inhibited tumor cell replication and blocked the stimulatory action of activin on cell growth. The activin action is likely mediated by specific receptors because cross-linking of [125]activin to the 50-55 kilodalton type I and 75-80 kilodalton type II receptors was found using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Northern blot analysis also revealed follistatin mRNA expression in the tumor cells, suggesting these cells are related to granulosa cells. Our findings indicate that activin can act as an autocrine growth factor in stimulating the proliferation of gonadal tumor cell lines derived from inhibin-alpha and p53-deficient mice and inhibits progesterone production. These tumor cell lines are useful for studies on the regulation of gonadal cell proliferation and steroidogenesis as well as the signaling pathway mediating activin action.

Cloning and functional expression of a rat brain corticotropin releasing factor (CRF) receptor.

Corticotropin releasing factor (CRF), a key neuroregulator of the hypothalamic-pituitary-adrenal cortical axis, also displays a broad range of effects on the endocrine, central nervous and immune systems. Having recently characterized the human pituitary CRF receptor by expression cloning of cDNA from a human Cushing's corticotropic adenoma, we report here the structure of the cDNA for a rat brain CRF receptor (rCRF-R) which was cloned by hybridization from a rat brain cDNA library. The sequence of the rCRF-R encodes a 415 amino acid protein comprising seven membrane spanning domains. The rCRF-R is 97% identical at the amino acid level to the human pituitary tumor CRF receptor, differing by only 12 amino acids. When expressed in COSM6 cells, the rCRF-R binds CRF with high affinity (Kd = 1.7 (0.8-3.8)nM). The receptor transduces a CRF stimulated accumulation of intracellular cAMP which is inhibited by the CRF antagonist, alpha helCRF(9-41). These results suggest that the brain expresses a CRF receptor similar to that in the pituitary.

Cloning and characterization of human urocortin.

Urocortin, a new member of the CRF peptide family which also includes urotensin I and sauvagine, was recently cloned from the rat midbrain. The synthetic replicate of urocortin was found to bind with high affinity to type 1 and type 2 CRF receptors and, based upon its anatomic localization within the brain, was proposed to be a natural ligand for the type 2 CRF receptors. Using a genomic library, we have cloned the human counterpart of rat urocortin and localized it to human chromosome 2. Human and rat urocortin share 95% identity within the mature peptide region. Synthetic human urocortin binds with high affinity to CRF receptor types 1, 2 alpha, and 2 beta, stimulates cAMP accumulation from cells stably transfected with these receptors, and acts in vitro to release ACTH from dispersed rat anterior pituitary cells. In addition, the CRF-binding protein binds human urocortin with high affinity and can prevent urocortin-stimulated ACTH secretion in vitro. The inhibitory effect of the CRF-binding protein on human urocortin can be blocked by biologically inactive CRF fragments, such as CRF(9-33).

Hybridization histochemical localization of activin receptor subtypes in rat brain, pituitary, ovary, and testis.

We have studied the distribution of activin receptor gene expression in the brain, pituitary, ovary, and testis of the adult rat by in situ hybridization, using probes complementary to the mRNAs encoding the mouse activin receptor subtypes II and IIB (ActRII and ActRIIB). Throughout the brain, ActRII mRNA expression was stronger than that of ActRIIB, and the patterns of expression were similar, although not identical. The most intense sites of activin receptor gene expression were the hippocampal formation, especially the dentate gyrus (ActRII), taenia tecta, and induseum griseum; the amygdala, particularly the amygdaloid-hippocampal transition zone; and throughout the cortical mantle, including the primary olfactory cortex (piriform cortex and olfactory tubercle); other regions of the cortex showing lesser degrees of hybridization included the cingulate cortex, claustrum, entorhinal cortex, and subiculum. In addition, moderate levels of expression were observed in several hypothalamic areas involved in neuroendocrine regulation, such as the suprachiasmatic, supraoptic, paraventricular, and arcuate nuclei. Moreover, activin receptors were also expressed in regions with inputs to the hypothalamus, both in the forebrain (bed nucleus of the stria terminalis and medial preoptic area) and within the brainstem (nucleus of the solitary tract, dorsal motor nucleus of the vagus, locus coeruleus, and mesencephalic raphé system). ActRII mRNA was observed in the intermediate lobe of the pituitary and, less prominently, in the anterior lobe, whereas ActRIIB appeared to be weakly expressed throughout all three pituitary divisions. In both male and female gonads, activin receptor message was clearly present in germ cells, and ActRII was the predominant form. In the ovary, in addition to an intense signal in the oocyte, activin receptor was expressed in corpus luteum and granulosa cells during diestrous day 1. In the testis, there was a strong ActRII signal in rounded spermatids, and a moderate signal in pachytene spermatocytes. In contrast, ActRIIB was absent within tubules, but weakly expressed in interstitial and Leydig cells. This is the first report of the distribution of activin receptor message in adult mammalian tissues. Although consistent with some previously suggested functional associations of activin-containing pathways in the brain, this pattern of expression suggests a greater role for activin than was previously appreciated in cortical, limbic, and somatosensory pathways and in the maturation of germ cells in the gonads of both male and female rats.

Avidin- or streptavidin-biotin as a highly sensitive method to stain total protein on membranes.

A sensitive method for staining proteins after transfer from polyacrylamide gels to nitrocellulose paper is described. Transferred proteins are first derivatized by reaction of the nitrocellulose replica with sulfosuccinimidobiotin and are then reacted sequentially with streptavidin, rabbit anti-streptavidin, and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. Incubation with the enzyme substrate alpha-chloronaphthol, produces dark protein bands against a white background. The binding of streptavidin to the proteins is dependent on biotin derivatization as demonstrated by competition with biotinylated bovine serum albumin or 10 nM biotin. The procedure detects less than 5 ng of transferred protein in a single band and is thus 5-10 times more sensitive than horseradish peroxidase-conjugated avidin alone. For bovine serum albumin, the method is comparable in sensitivity to silver staining of protein in polyacrylamide gels.

Stereochemistry-dependent inhibition of RAS farnesylation by farnesyl phosphonic acids.

This investigation compares the effects of three farnesyl pyrophosphate analogs on selected aspects of isoprenoid metabolism. E,E-alpha-Hydroxyfarnesylphosphonate was prepared by an improved variation on a literature synthesis, which also gave access to the new Z,E-alpha-hydroxyfarnesyl- and alpha-hydroxygeranylphosphonates. A striking find is that only E,E-alpha-hydroxyfarnesylphosphonate induces alteration of RAS processing in intact human-derived leukemia cells and inhibits farnesyl protein transferase in enzyme assays, while the Z,E-alpha-farnesyl- and geranylphosphonates are inactive. The inhibitory activity of E,E-alpha-hydroxyfarnesylphosphonate is greater in enzyme than intact cell assays. This active compound does not significantly inhibit geranylgeranyl protein transferase I or squalene synthase, nor does it diminish cholesterol synthesis. These results indicate that the length of the terpenoid chain and olefin stereochemistry allow selective inhibition of critical enzymes of terpenoid metabolism. Discrimination was observed between inhibition of farnesyl protein transferase and squalene synthase by E,E-alpha-hydroxyfarnesylphosphonate, even though both enzymes utilize farnesyl pyrophosphate as their natural substrate.

Ankle-joint salvage following a tibial pilon fracture. A case report.

This article presents a case of a tibial pilon fracture following a motor-vehicle accident. It discusses the main classification system and mechanism of injury for such fractures and emphasizes an alternative form of treatment of the usually suggested ankle fusion: an arthrectomy, which allows motion, thereby salvaging the ankle joint.

Monoclonal antibodies against Muscleblind-like 3, a protein with punctate nuclear localization.

Muscleblind-like 3 (MBNL3) belongs to a family of RNA binding proteins that regulate alternative splicing. We have generated a set of monoclonal antibodies (MAbs) against mouse MBNL3, three of which do not cross-react with the other muscleblind-like (MBNL) proteins, MBNL1 and MBNL2. Epitope mapping revealed that MAbs P1C7, P1E7, SP1C2, and P2E6 recognize distinct, non-overlapping segments of the MBNL3 polypeptide sequence. Immunohistochemical staining of proliferating muscle precursor cells localized MBNL3 to the nucleus in a punctate pattern, characteristic of subcellular structures in the nucleus enriched in pre-messenger RNA splicing factors. Although MBNL3 did not co-localize with SC35 and PSP1 (widely used markers of splicing speckles and paraspeckles), the punctate localization pattern of MBNL3 within interchromatin regions of the nucleus is highly predictive of proteins involved in pre-mRNA processing. Monoclonal antibodies specific for mouse MBNL3 will facilitate further investigation of the expression pattern and unique functions of this splicing factor during development and in different adult mouse tissues.

Systematic review of ethnic variation in the phenotype of Behcet's disease.

OBJECTIVE: To systematically review the strength of evidence that supports the premise that there are different expressions in the clinical phenotype of Behcet's disease (BD) in different ethnic groups. METHODS: The hierarchy of evidence and inclusion criteria were decided prior to the search for relevant literature. We searched Medline and Embase databases between 1966 and March 2005 for publications related to epidemiology of Behcet's disease or syndrome. Relevant papers were extracted in hard copy and the references of all these papers were then hand searched for further articles. RESULTS: Four population-based studies, of which two were from Turkey, and seven non-population-based comparative studies were found. The majority of literature identified were case series and were not included in the review. There were insufficient cross-sectional studies from different populations to be able to compare phenotypic differences. None of the comparative studies found evidence of a difference in the clinical expression of BD between ethnic groups. CONCLUSIONS: There is insufficient evidence to support the premise that there are different expressions in the clinical phenotype of BD in different ethnic groups. Population-based, cross-sectional surveys or case-control studies using standardized criteria and clear ethnic definitions are suggested to investigate this hypothesis further.

Physical mapping of complex genomes by cosmid multiplex analysis.

A rapid and powerful approach for linking individual clones of a cosmid library and the assembly of a large physical map is presented, which depends on the simultaneous analysis of many cosmid clones for overlapping regions. This method uses cosmid vectors that contain endogenous bacteriophage T3 and T7 promoters to allow for the identification of overlapping clones through the synthesis of end-specific RNA probes. A genomic library is constructed and organized as an ordered matrix such that each clone is assigned an identifying coordinate. DNA from mixtures of cosmid clones is pooled such that each pool contains only one common member with any other pool, RNA probes are prepared from mixtures of cosmid clones, and groups of clones overlapping with the constituents of the mixtures are determined by hybridization. Pooled probes are most simply prepared by grouping clones according to the rows and columns of the library matrix. The pairwise comparison of data generated by the hybridization of mixed probes can be decoded by using simple algorithms that predict the order and linkage of all clones in the collection and organize them into predicted contigs. To demonstrate the feasibility of multiplexed analysis of cosmids, a genomic library was prepared from a mouse-human somatic cell hybrid that contains a portion of the long arm of human chromosome 11. Preparation, arrangement on a matrix, and analysis of pooled cosmid clones from this collection resulted in the detection of 1099 linked pairs of cosmids, which could be assembled into 315 contigs. Thus, with a minimal amount of effort, a substantial portion of this genomic region has been linked in multiple overlapping contigs. This method may have practical applications in the large-scale mapping and sequencing of mammalian genomes.

Changes in brain gonadotropin-releasing hormone, pituitary and plasma gonadotropins, and plasma thyroxine during smoltification in chinook salmon (Oncorhynchus tschawytscha).

Concentrations of brain salmon gonadotropin-releasing hormone (sGnRH), plasma gonadotropin I (GTH I), and pituitary GTH I and GTH II were determined in yearling chinook salmon (Oncorhynchus tschawytscha) during the parr-smolt transformation in two successive seasons. There were significant elevations in brain sGnRH content from February to March in 1988, and from February to April in 1989. Increases in brain sGnRH content coincided with elevations in plasma thyroxine levels that occurred from February to March, 1988 and 1989. Plasma GTH levels were relatively constant (1-2 ng/ml) throughout the period of sampling. However, during 1988, plasma concentrations of GTH I decreased significantly between late March and early April. During 1989, plasma GTH I levels appeared to reach a peak (2 ng/ml) in mid-February, but otherwise remained near 1 ng/ml. Previous studies have shown that GTH II was not detectable in plasma at this stage. During 1989, pituitary GTH I concentrations were 50- to 70-fold higher than that of GTH II, and increased, though not significantly, from February through April. Although GTH II was detected in the pituitary by RIA, it is likely that the measurable levels are due to GTH I cross-reaction in the GTH II RIA. Histological examination of the gonads indicated that throughout smoltification the oocytes remained in the perinucleolar stage of oogenesis and the testes were in the spermatogonial stage of spermatogenesis. Although no observable changes in gametogenesis occurred, the changes in brain sGnRH content, plasma GTH I levels, and pituitary GTH content suggest that some changes in the hypothalamic-pituitary axis may occur during smoltification.

Molecular organization of the human CD3 gene family on chromosome 11q23.

The genes encoding three invariant components of the human T-cell antigen receptor, the CD3 delta, gamma, and epsilon chains, are located on human chromosome 11 at band q23. We isolated cosmid clones containing the human CD3 delta and gamma chain genes in vectors designed for rapid and efficient chromosome "walking". The human CD3 epsilon gene was located in the region immediately downstream of the CD3 delta and gamma genes using synthetic oligonucleotide probes and the localization of this gene confirmed by DNA sequencing. Detailed restriction mapping of the CD3 locus demonstrated that all three CD3 subunits are encoded within 60 kb of DNA with the CD3 epsilon gene located 26 kb downstream of the CD3 delta and gamma genes. Analysis of genomic DNA on pulsed field gels using probes isolated from these cosmid clones defined a physical map of 750 kb spanning the CD3 locus on human chromosome 11q23. The CD3 genes thus comprise a multigene family encoding cell surface components important for transmembrane signaling on T lymphocytes. The arrangement of these genes suggest that they may share common regulatory elements for the control of gene expression during T-cell ontogeny.

Molecular localization of the t(11;22)(q24;q12) translocation of Ewing sarcoma by chromosomal in situ suppression hybridization.

Chromosome translocations are associated with a variety of human leukemias, lymphomas, and solid tumors. To localize molecular markers flanking the t(11;22) (q24;q12) breakpoint that occurs in virtually all cases of Ewing sarcoma and peripheral neuroepithelioma, high-resolution chromosomal in situ suppression hybridization was carried out using a panel of cosmid clones localized and ordered on chromosome 11q. The location of the Ewing sarcoma translocation breakpoint was determined relative to the nearest two cosmid markers on 11q, clones 23.2 and 5.8, through the analysis of metaphase chromosome hybridization. By in situ hybridization to interphase nuclei, the approximate physical separation of these two markers was determined. In both Ewing sarcoma and peripheral neuroepithelioma, cosmid clone 5.8 is translocated from chromosome 11q24 to the derivative chromosome 22 and a portion of chromosome 22q12 carrying the leukemia inhibitory factor gene is translocated to the derivative chromosome 11. The physical distance between the flanking cosmid markers on chromosome 11 was determined to be in the range of 1000 kilobases, and genomic analysis using pulsed-field gel electrophoresis showed no abnormalities over a region of 650 kilobases in the vicinity of the leukemia inhibitory factor gene on chromosome 22. This approach localizes the Ewing sarcoma breakpoint to a small region on chromosome 11q24 and provides a rapid and precise technique for the molecular characterization of chromosomal aberrations.