Estimation of tile drainage contribution to streamflow and nutrient loads at the watershed scale based on continuously monitored data.

Nitrogen losses from artificially drained watersheds degrade water quality at local and regional scales. In this study, we used an end-member mixing analysis (EMMA) together with high temporal resolution water quality and streamflow data collected in the 122 km2 Otter Creek watershed located in northeast Iowa. We estimated the contribution of three end-members (groundwater, tile drainage, and quick flow) to streamflow and nitrogen loads and tested several combinations of possible nitrate concentrations for the end-members. Results indicated that subsurface tile drainage is responsible for at least 50% of the watershed nitrogen load between April 15 and November 1, 2015. Tiles delivered up to 80% of the stream N load while providing only 15-43% of the streamflow, whereas quick flows only marginally contributed to N loading. Data collected offer guidance about areas of the watershed that should be targeted for nitrogen export mitigation strategies.

The first confirmed cases of full albinism in rajid species.

Three albino skate specimens (Rajidae) were captured from the North Sea and English Channel between 2008 and 2011. Using DNA barcoding (COI gene) and morphometric analyses, species were identified as a spotted ray Raja montagui, a blonde ray Raja brachyura and a thornback ray Raja clavata. This finding represents the first record of full albinism (a lack of skin and retinal pigmentation) in rajid species.

Low genetic diversity in a snail intermediate host (Biomphalaria pfeifferi Krass, 1848) and schistosomiasis transmission in the Senegal River Basin.

Population genetic perturbations of intermediate hosts, often a consequence of human pressure on environmental resources, can precipitate unexpectedly severe disease outbreaks. Such disturbances are set to become increasingly common following range changes concomitant with climate shifts, dwindling natural resources and major infrastructure changes such as hydroprojects. Construction of the Diama dam in the Senegal River Basin (SRB) reduced river salinity, enabling the freshwater snail intermediate host Biomphalaria pfeifferi to rapidly expand its distribution. A serious public health problem ensued, with an epidemic of intestinal schistosomiasis occurring in the previously schistosome-free Richard-Toll region within 2 years. The current study aimed to assess the population variability of B. pfeifferi in the SRB, and speculate upon its subsequent impact on host-parasite interactions following such engineered ecological change. Genetic variation at nine polymorphic microsatellite loci revealed little population differentiation in SRB snails compared with those from natural habitats in Zimbabwe, where Schistosoma mansoni transmission is much lower. 'Open' SRB habitats are associated with greater water contact, smaller population sizes and less genetic diversity, with sites downstream of Richard-Toll showing greater inter- and intrapopulation variation, concomitant with less frequent human contact. These observations may be explained by rapid expansion into pristine habitat selecting for high fecundity genotypes at the expense of schistosome resistance, presenting S. mansoni with genetically homogenous highly fecund susceptible populations around the focal point, promoting development of a highly compatible host-parasite relationship. Longitudinal study of such systems may prove important in predicting public health risks engendered by future environmental engineering projects.

Radioimmunoassay for abnormal hemoglobins.

A sensitive and specific radioimmunoassay has been developed for the identification or quantification of the human hemoglobin variants S, C, D-Los Angeles, E, G Philadelphia, Russ, O Arab, Beograd, J Paris I, G San Jose, Q Iran, Korle Bu, and F Malta I. In the immunoassay, monospecific antibody preparations are used which recognize the single amino acid substitution in the variant polypeptide chail and do not cross-react with normal hemoglobins or hemoglobin variants containing a different amino acid exchange at the same position.

Size-dependent growth of individual Atlantic salmon Salmo salar alevins from hatch to first feeding.

Variation in egg size, hatch timing and size at hatch, and their influence on individual growth rates of Atlantic salmon Salmo salar alevins up to first feeding were examined in pure strain and hybrid crosses of fish from Scotland and Canada. At the intra-female, intra-cross type and inter-cross type levels, specific growth rates prior to first feed were strongly size dependent, with smaller and later hatching alevins growing significantly faster. The magnitude of this size-dependent growth was greatest in the hybrid crosses. This resulted in a 40% reduction in the coefficient of variation (c.v.) in alevin size from post-hatch to first feeding at the intra-female level, and a reduction of both intra- and inter-cross differences in alevin sizes in the same period.

The effects of lubrication on the temperature rise and surface finish of amalgam and composite resin.

UNLABELLED: It was thought that when finishing and polishing direct filling materials lubrication would affect the surface roughness and temperature rise in samples of amalgam and composite. OBJECT: Previous work by the authors has shown that there is an optimum load, speed and time that produced the smoothest surface when finishing amalgam and composite resin using each of four grades of a disc system. This work was undertaken to examine the effects on temperature rise in samples of amalgam and composite resin of finishing dry compared to finishing with different lubricants. The experiments all used these optimum loads, speeds and times. It also compares the surface finish produced using different lubricants. MATERIALS AND METHODS: A high copper amalgam and a hybrid composite resin were finished using the four grades of abrasive discs. Samples produced were 25 mm long by 6 mm wide by 2 mm deep. A thermocouple was inserted 1 mm into the base of the samples. The thermocouple was connected via an electronic thermometer to a computer that permitted the display and recording of temperature against time. After roughening, the samples were finished and polished in a specially constructed jig that mimicked oral finishing. The pre-determined optimum loads, speeds and times were used sequentially for each of the four grades of disc. Five samples were tested for each method of finishing. Firstly, run dry, then in turn lubricated with water, walnut oil and petroleum jelly. After the use of each abrasive disc the surface roughness was measured. One of the five samples was selected at random and prepared for examination in the scanning electron microscope. All results were subjected to non-parametric statistically analyses. RESULTS: With both materials the temperature rise was greatest when run dry, followed by petroleum jelly, walnut oil and the least was when lubricated with water. With these two materials the surface roughness correlates negatively with the temperature rise. The smoothest surface being achieved when finished dry. CONCLUSIONS: To obtain the smoothest surface finish amalgam and composite should be finished dry but further work is needed to assess the effect of the temperature rise found in the materials on the pulp.

The effects of lubrication on the temperature rise and surface finish of glass-ionomer cements.

OBJECT: Previous work [Jones CS. Factors influencing the finishing of direct filling materials. PhD Thesis, University of London; 2002] has shown that there is an optimum load, speed and time that produced the smoothest surface when finishing glass-ionomer cement using each of four grades of a disc system. This study looks at the effects of lubrication on the temperature produced in samples of GIC when finished dry and with different lubricants using these optimal loads, speeds and times. It also compares the surface finish produced using different lubricants. MATERIALS AND METHODS: A thermocouple connected so that it permitted the display and recording of temperature against time was inserted 1mm into the base of samples of a glass-ionomer cement. The samples were finished and polished using each of the grades of a disc system in a specially constructed jig that mimicked oral finishing. After roughening, the pre-determined optimum loads, speeds and times were used sequentially for each of the four grades of disc. Five samples were tested for each method of finishing. Firstly run dry, then in turn lubricated with water, walnut oil and petroleum jelly. After the use of each abrasive disc the surface roughness was measured using a profilometer. One of the five samples was selected at random and prepared for examination in the scanning electron microscope. All results were subjected to non-parametric statistically analyses. RESULTS: Walnut oil and petroleum jelly produced significant temperature increases compared to both dry and with water finishing. Lubricated with water significantly reduced the temperature rise compared to dry. The Ra values of 0.5 microm was obtained for the coarse and a value of 0.3 microm for the medium discs run without lubrication. With lubrication the Ra increased although there was little difference between the lubricants. However the photomicrographs showed that walnut oil and petroleum jelly caused gross morphological changes indicating major surface destruction. CONCLUSIONS: The practice of finishing GICs using petroleum jelly or similar lubricant appears to be detrimental. Further experimental work needs to be done to advise practitioners on finishing GICs to produce the smoothest surface possible.

The H-ras oncogene interferes with retinoic acid signaling and metabolism in NIH3T3 cells.

We have previously shown that retinoic acid (RA) fails to induce transglutaminase C in H-ras transformed NIH-3T3 cells. Therefore, we investigated the effect of the H-ras oncogene on the metabolism of RA and on the expression of the cellular RA-binding protein I mRNA. HPLC analysis of the media and cell extracts demonstrated that H-ras-transformed cells metabolize RA to a much lesser extent than control cells, resulting in a higher concentration of RA in H-ras cells. Although inactive in endogenous transglutaminase induction, H-ras cell-associated RA was shown to be biologically available to induce activation of a reporter construct containing a retinoid response element and in stimulating transglutaminase activity in nontransfected cells. Cellular RA-binding protein I mRNA, supposedly involved in RA storage, was significantly increased in the H-ras-transformed cells. These data demonstrate that, even though H-ras-transformed cells accumulate up to 20 fold the concentration of RA as NIH-3T3 cells, they fail to show transglutaminase induction, suggesting that H-ras interferes with signal transduction by RA.

Retinyl palmitate, retinyl phosphate, and dolichyl phosphate of postnuclear membrane fraction from hepatoma, host liver, and regenerating liver: marginal vitamin A status of hepatoma tissue.

The retinyl palmitate content of the postnuclear membrane fraction from 10 Morris hepatomas, their host rat livers, one acetylaminofluorene-induced rat liver hepatoma, and the host liver and of regenerating rat liver was measured by reverse-phase high-pressure liquid chromatography of the chloroform:methanol extracts. Membranes from the hepatoma tissue contained less than detectable levels of retinyl acyl esters, whereas membranes from host liver tissue and regenerating liver contained levels of retinyl palmitate within normal ranges. The amount of cellular retinol-binding protein was also decreased considerably in cytosols from 9618 and 7777 hepatomas. The ratio of endogenous retinyl phosphate to the polyisoprenoid dolichyl phosphate available for mannosylation in an assay containing postnuclear membranes and guanosine dephospho[14C] mannose was decreased by a factor of 3 to 10 in hepatoma tissue. Such change in ratio was not attributable to specific changes in retinyl phosphate mannose-synthesizing activity, but it appeared to be related to the vitamin A deficiency condition of the membrane from tumors. As for membranes from vitamin A-deficient liver tissue, postnuclear membranes from rat cystic hepatocarcinoma, Morris 7777, 3924A1-1, and 5123D-1-2 transplantable rat hepatomas and guinea pig line 10 hepatoma all synthesized a mannolipid with intermediate hydrophobic properties between retinyl phosphate mannose and dolichyl phosphate mannose and not normally found in liver tissue. These alterations in patterns of lipid intermediates may be responsible for altered glycosylation of glycoproteins in neoplastic cells. In conclusion, the present investigation establishes that hepatoma cell membrane is in a status of vitamin A and of retinyl phosphate depletion, while dolichyl phosphate contents appear similar to host liver membrane.

Retinoic acid increases tyrosine phosphorylation of focal adhesion kinase and paxillin in MCF-7 human breast cancer cells.

Treatment of estrogen receptor (ER)-positive MCF-7 human breast cancer cells with retinoic acid (RA) inhibited cell growth and increased cell adhesion to fibronectin. In contrast, ER- MDA-MB-231 cells failed to respond. Western blot analysis showed that tyrosine phosphorylation of two major bands at Mr 125,000 and Mr 68,000 was induced by RA in ER+ MCF-7 human breast carcinoma cells. However, this induction was a late phenomenon detectable at 12 and 24 h, but not within 3 h. A similar increase of tyrosine phosphorylation by RA was observed in ER+ human breast cancer cell lines T-47D and ZR-75-1, but not in the ER- cell lines MDA-MB-231, MDA-MB-453, and MDA-MB-468. Focal adhesion kinase and paxillin, which localize in focal adhesion plaques and may play important roles in the integrin signaling pathway, were identified as the major proteins showing RA-induced tyrosine phosphorylation. The retinoid X receptor-selective compound SR11237 failed to induce tyrosine phosphorylation, indicating that retinoid X receptor activation is not involved in this phenomenon. In contrast, stable overexpression of a truncated RA receptor (RAR) alpha cDNA, RARalpha403, with strong RAR dominant negative activity prevented the increase in tyrosine phosphate, suggesting that RAR signaling is involved in RA-induced tyrosine phosphorylation. Tyrosine phosphorylation was induced the most by the RAR-alpha (193836), followed by RAR-gamma (194433), but was not significantly induced by RAR-gamma (193174)-selective retinoids. This study demonstrates a coordinated albeit relatively late effect of RA on cell adhesion and tyrosine phosphorylation in ER+ human breast cancer cells and suggests RAR-alpha as the major responsible retinoid receptor.

Biochemical and growth inhibition studies of methotrexate and aminopterin analogues containing a tetrazole ring in place of the gamma-carboxyl group.

The biological activities of novel analogues of methotrexate (MTX) and aminopterin (AMT) in which the gamma-carboxyl was replaced by a 1H-tetrazol-5-yl ring, an isosteric group with acidic properties similar to a carboxyl group, were investigated. The tetrazolyl analogues of MTX and AMT were more potent inhibitors of the growth of CCRF-CEM and K562 human leukemia cell lines during continuous (120 h) and 24-h pulse exposure than were the respective parent drugs; only when the exposure time was reduced to 6 h were the parent drugs more potent. These inhibitory effects on growth correlated with the onset of and recovery from inhibition of de novo thymidylate biosynthesis. Growth inhibition by the analogues was protectable by leucovorin. MTX-resistant CCRF-CEM sublines with decreased transport or increased dihydrofolate reductase (DHFR) levels were cross-resistant to the analogues. The analogues were as potent as their parent drugs in inhibiting DHFR activity in vitro and at displacing [3H]MTX from intracellular DHFR. Each analogue was more effective than its parent drug at inhibiting uptake of [3H]MTX into CCRF-CEM cells. The tetrazole analogue of AMT was a linear competitive inhibitor (Kis = 50 microM) of CCRF-CEM folylpolyglutamate synthetase, while the tetrazole analogue of MTX, unlike all other inhibitors, was linear noncompetitive (Kis = 51 microM, Kii = 321 microM). The data suggest that, compared with MTX or AMT, the tetrazole substituent, in place of the gamma-carboxyl group, allows more efficient transport into cells via the reduced folate/MTX carrier and the resulting greater uptake of the analogues leads to inhibition of DNA synthesis and cell death at lower extracellular concentrations during long exposures. The mechanism of cell death could involve inhibition at folypolyglutamate synthetase, but DHFR is the primary target. The low potency of the analogues during short exposure is presumably related to the inability to form the poly-gamma-glutamyl metabolites required for intracellular retention.

Enzymatic hydrolysis of sphingomyelin liposomes by normal tissues and tissues from patients with Niemann-Pick disease.

Liposomes of [3H]sphingomyelin are readily hydrolyzed by extracts of human spleen, liver, cultured skin fibroblasts and purified placental sphingomyelinase in the absence of detergents. The pH optimum for hydrolysis by liver and spleen extracts was 6.5-7.0 while the fibroblast activity showed an optimum at pH 4.0-4.3. However, the pH optimum for purified placental sphingomyelinase in the presence of Triton X-100 (pH 5.0) is only slightly different from that displayed with liposomes (pH 5.3). The data clearly show that hydrolysis of liposomal sphingomyelin by sphingomyelinase is affected by the composition and purity of the enzyme source.

Complex kinetics of bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine hydrolysis by purified sphingomyelinase in the presence of Triton X-100.

We have examined the hydrolysis of the synthetic phosphodiesters, bis(4-methylumbelliferyl)phosphate and hexadecanoyl(nitrophenyl)phosphorylcholine, by purified placental sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12) in the presence of Triton X-100. Triton X-100 enhanced activity with bis(4MU)phosphate at all concentrations tested. At very low concentrations of detergent, bis(4MU)phosphate hydrolysis approached zero. Our results indicate that bis(4MU)phosphate does not form a micelle with Triton X-100. The observed enhancement of bis(4MU)phosphate activity with Triton X-100 is likely due to a direct effect of detergent on the enzyme itself. HDNP-phosphorylcholine formed its own micelle (or liposome) in the absence of Triton X-100 and, at substrate concentrations below 4 mM, hydrolysis was inhibited by Triton X-100. The extent of this inhibition varied with detergent concentrations but could be totally eliminated at substrate values above 4 mM. For theoretical reasons kinetic constants which could be obtained with the HDNP-phosphorylcholine substrate at concentrations above 4 mM are not considered to be truly representative of the real values. We conclude that neither substrate is recommended to describe the true kinetic parameters pertaining to purified sphingomyelinase. In addition, bis(4MU)phosphate may not be suitable as an aid for diagnosis of sphingomyelinase deficiency states.U

Interaction of free porphyrins and metalloporphyrins with mouse ferrochelatase. A model for the active site of ferrochelatase.

The ability of purified mouse ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1) to bind and catalytically utilize a variety of porphyrins has been examined. In all, the kd, Km or Ki values for eleven different porphyrins, the Ki values for four metalloporphyrins and the kd values for two metalloporphyrins were determined. The data obtained demonstrate that mouse ferrochelatase binds a wide variety of porphyrins and metalloporphyrins with kd values ranging from 6 nM for N-methylprotoporphyrin to 1.08 microM for coproporphyrin III. However, the enzyme shows a degree of catalytic specificity for the substituents at the 2.4 positions and utilizes only proto-, hemato-, meso-, deutero-, 2,4-monohydroxy-ethylmonovinyl- and 2,4-monohydroxymethylmonovinyl deuteroporphyrin as substrates. The data show that the magnitude of the kd is not an accurate indicator of the ability of the porphyrin to serve as a substrate or inhibitor and, with the exception of N-methylprotoporphyrin, the size of the kd is several orders of magnitude less than that of the Km or Ki. Of the metalloporphyrins examined (Fe, Co, Zn and Sn) all inhibited ferrochelatase at micromolar concentrations, although tin protoporphyrin was the least effective. These data are discussed in terms of an active site model for mammalian ferrochelatase.

Biosynthetic studies on mannolipids and mannoproteins of normal and vitamin A-depleted hamster livers.

The incorporation of [1-14C]mannose into hamster liver glycolipids and glycoproteins was studied in normal and vitamin A-depleted hamsters. Severly (25% weight loss) and mildly (no weight loss) deficient animals were compared to vitamin A-fed controls. The incorporation of [14C]mannose into glycolipids and glycoproteins decreased in mild and severe vitamin A deficiency by 63-90% compared to vitamin A-fed animals. These results were essentially the same whether expressed per g of wet liver, per DNA or per protein. The size of the pools of mannose, glucose and galactose and their specific radioactivity in liver were determined by gas-liquid chromatography of the boronates of the hexitols (Eisenberg, Jr, F. (1972) Methods Enzymol. XXVIIIB, 168-178) in normal and vitamin A-deficient conditions. It was found that the amount of free hexoses per g of liver was very similar in normal and vitamin A-deficient conditions. The specific radioactivities for mannose and glucose were greater in vitamin A deficiency, thus excluding the possibility that the observed severe decrease in glycopeptide and glycolipid synthesis is a reflection of a similar decrease in the specific radioactivity of the precursor pools. Quantitation of mannose in glycoprotein showed a 79% decrease in vitamin A deficiency. Specific radioactivity of mannose in glycoproteins, 20 min after injection of the label, was 187 dpm/mug of mannose in the normal and 48 kpm/mug of mannose in the vitamin A-deficient livers. It is concluded that vitamin A is necessary for the biosynthesis of liver mannose-containing glycoproteins and glycolipids.

Comparison of the uptake and metabolism of retinol delivered to primary mouse keratinocytes either free or bound to rat serum retinol-binding protein.

Serum retinol-binding protein (RBP) is believed to be responsible for the transport of retinol from its storage site in the liver to vitamin A requiring target cells such as keratinocytes. We have used primary mouse keratinocytes as a model system to compare the uptake and metabolism of [3H] retinol delivered to them either free in solution or bound to RBP. RBP was purified from rat serum, loaded with [3H]retinol, and the [3H]retinol-RBP complex purified by affinity chromatography on human transthyretin-Sepharose. Keratinocytes incubated with either free [3H]retinol or [3H]retinol-RBP complex accumulated [3H]retinol in a time and temperature dependent manner. However, cells incubated with free [3H]retinol acquired 15- to 20-fold more ligand than if the retinol was delivered via RBP. The uptake of free [3H]retinol or [3H]retinol from RBP was not inhibited by excess unlabeled free retinol. The uptake of [3H]retinol from RBP was inhibited by high concentrations of holo-RBP, with half maximal inhibition occurring at 3 microM holo-RBP. However, no specific binding of 125I-labeled RBP to monolayers of keratinocytes or membranes prepared from them was found indicating the absence of a high affinity RBP receptor on keratinocytes. Surprisingly, 50% of the [3H]retinol delivered to the keratinocytes during a 30-min uptake period was released from them within 30-min irrespective of whether or not it was initially delivered to them as free [3H]retinol or bound to RBP. The remaining 50% was lost at a much slower rate, but only 20% remained 24-h after delivery. Studies on retinol metabolism demonstrated that 7%-12% of the total cell-associated [3H]retinol delivered during a 90-min uptake period was esterified (mostly as retinyl palmitate) whether or not it was given free in solution or bound to RBP. Additionally, [3H]retinol taken up by the keratinocytes during the initial 90-min incubation was not chased into a stable retinyl ester pool in a subsequent 9.5-h incubation, but instead, retinyl ester was lost from the cells with kinetics similar to those of total cell-associated radioactivity. These results suggest that a function of RBP is to protect cells from a rapid accumulation of the vitamin which occurs when it is delivered free in solution. However, the cellular fate and metabolism of retinol appears to be the same whether the vitamin is delivered free in solution or bound to RBP.

Conversion of murine Fabs isolated from a combinatorial phage display library to full length immunoglobulins.

The use of combinatorial Ig libraries displayed on the surface of bacteriophage has advantages over traditional hybridoma techniques for the generation of mAbs but in many instances full length Igs may be more desirable than Fab fragments. Two murine Fabs reactive with the human complement component C5a, recovered from a combinatorial library, were converted to full length IgG2a mAbs. The VH and VL domains of these antibodies were removed from the bacterial expression vector used for the combinatorial library construction, and subcloned into individual mammalian expression vectors containing the corresponding Ig heavy and light chain constant regions. The subcloning relied on 5' restriction endonuclease sites encoded by the oligonucleotide primers originally used to amplify the Ig cDNAs and 3' sites conserved in CH1 and C kappa. These vectors were co-transfected into COS cells yielding full length IgG2a versions of the anti-C5a antibodies. The mAbs, purified from the culture supernatant, retained the full activity of the Fabs, binding specifically to and neutralizing human recombinant C5a. Refined versions of the mammalian expression vectors have been constructed for single step conversion of murine recombinant Fabs, recovered from combinatorial libraries, to IgG2a mAbs.

Structure-based design of cathepsin K inhibitors containing a benzyloxy-substituted benzoyl peptidomimetic.

Peptidomimetic cathepsin K inhibitors have been designed using binding models which were based on the X-ray crystal structure of an amino acid-based, active site-spanning inhibitor complexed with cathepsin K. These inhibitors, which contain a benzyloxybenzoyl group in place of a Cbz-leucine moiety, maintained good inhibitory potency relative to the amino acid-based inhibitor, and the binding models were found to be very predictive of relative inhibitor potency. The binding mode of one of the inhibitors was confirmed by X-ray crystallography, and the crystallographically determined structure is in close qualitative agreement with the initial binding model. These results strengthen the validity of a strategy involving iterative cycles of structure-based design, inhibitor synthesis and evaluation, and crystallographic structure determination for the discovery of peptidomimetic inhibitors.