The impacts of cattle access points on deposited sediment levels in headwater streams in Ireland

Cattle access to streams has been linked globally with degradation of stream water quality, driven largely by bank erosion and resultant instream, fine sediment deposition. The majority of evidence on such effects is however based in arid and semiarid regions of the United States and Australia, with few studies relating to cool temperate climates such as Northwest Europe. In this study, “Quorer” resuspendable sediment samples were taken from riffle geomorphic units upstream (control) and at two points downstream (pressure and recovery) of cattle access points in headwater streams in agricultural catchments in Ireland to assess levels of deposited stream sediment. Samples were taken in April/May (2016) prior to the grazing season and in October (2016) at the end of the grazing season. Sites in good‐high ecological status catchments and less than good ecological status catchments were included in the study. Higher levels of sediment were found downstream of cattle access points in both good‐high status and less than good status catchments; however, the impacts of access points were spatially confined to, in most cases, the area immediately downstream of the point of access. There was a strong correlation between deposited sediment mass and organic matter (OM) mass, with levels of OM increasing linearly with deposited sediment mass. Levels of measured sediment were negatively correlated with riparian habitat health (measured using a qualitative habitat assessment). The results of this study highlight the need for measures to prevent cattle access to headwater streams where access points can be many in order to manage local habitat quality and downstream water quality issues.     

Characterizing physical habitat preferences and thermal refuge occupancy of brook trout (Salvelinus fontinalis) and Atlantic salmon (Salmo salar) at high river temperatures

Anthropogenic influences, including climate change, are increasing river temperatures in northern and temperate regions and threatening the thermal habitats of native salmonids. When river temperatures exceed the tolerance levels of brook trout and Atlantic salmon, individuals exhibit behavioural thermoregulation by seeking out cold‐water refugia – often created by tributaries and groundwater discharge. Thermal infrared (TIR) imagery was used to map cold‐water anomalies along a 53 km reach of the Cains River, New Brunswick. Trout and salmon parr did not use all identified thermal anomalies as refugia during higher river temperature periods (>21°C). Most small‐bodied trout (8–30 cm) were observed in 80% of the thermal anomalies sampled. Large‐bodied trout (>35 cm) required a more specific set of physical habitat conditions for suitable refugia, that is, 100% of observed large trout used 30% of the anomalies sampled and required water depths >65 cm within or adjacent to the anomaly. Densities of trout were significantly higher within anomalies compared with areas of ambient river temperature. Salmon parr were less aligned with thermal anomalies at the observed temperatures, that is, 59% were found in 65% of the sampled anomalies; and densities were not significantly different within/ outside anomalies. Salmon parr appeared to aggregate at 27°C, and after several events over 27°C variability in aggregation behaviour was observed – some fish aggregated at 25°C, others did not. We stipulate this is due to variances of thermal fatigue. Habitat suitability curves were developed for velocity, temperature, depth, substrate, and deep water availability to characterize conditions preferred by fish during high‐temperature events. These findings are useful for managers as our climate warms, and can potentially be used as a tool to help conserve and enhance thermal refugia for brook trout and Atlantic salmon in similar systems.     

Traumatic vertebral arterial dissection and vertebrobasilar arterial thrombosis successfully treated with endovascular thrombolysis and stenting

A case of traumatic extracranial vertebral arterial dissection leading to vertebrobasilar thrombosis and respiratory compromise requiring mechanical ventilation was managed with intraarterial thrombolysis and stenting of the vertebral intimal dissection. In contrast to similar, previously reported cases, this critically ill patient made a full recovery, returning to his job as a secondary school teacher.     

Identification of in vitro phosphorylation sites in the growth cone protein SCG10. Effect Of phosphorylation site mutants on microtubule-destabilizing activity

SCG10 is a neuron-specific, membrane-associated protein that is highly concentrated in growth cones of developing neurons. Previous studies have suggested that it is a regulator of microtubule dynamics and that it may influence microtubule polymerization in growth cones. Here, we demonstrate that in vivo, SCG10 exists in both phosphorylated and unphosphorylated forms. By two-dimensional gel electrophoresis, two phosphoisoforms were detected in neonatal rat brain. Using in vitro phosphorylated recombinant protein, four phosphorylation sites were identified in the SCG10 sequence. Ser-50 and Ser-97 were the target sites for protein kinase A, Ser-62 and Ser-73 for mitogen-activated protein kinase and Ser-73 for cyclin-dependent kinase. We also show that overexpression of SCG10 induces a disruption of the microtubule network in COS-7 cells. By expressing different phosphorylation site mutants, we have dissected the roles of the individual phosphorylation sites in regulating its microtubule-destabilizing activity. We show that nonphosphorylatable mutants have increased activity, whereas mutants in which phosphorylation is mimicked by serine-to-aspartate substitutions have decreased activity. These data suggest that the microtubule-destabilizing activity of SCG10 is regulated by phosphorylation, and that SCG10 may link signal transduction of growth or guidance cues involving serine/threonine protein kinases to alterations of microtubule dynamics in the growth cone.     

A noncompetitive peptide inhibitor of the nicotinic acetylcholine receptor from Conus purpurascens venom

A paralytic peptide, psi-conotoxin Piiie has been purified and characterized from Conus purpurascens venom. Electrophysiological studies indicate that the peptide inhibits the nicotinic acetylcholine receptor (nAChR). However, the peptide does not block the binding of alpha-bungarotoxin, a competitive nAChR antagonist. Thus, psi-conotoxin Piiie appears to inhibit the receptor at a site other than the acetylcholine-binding site. As ascertained by sequence analysis, mass spectrometry, and chemical synthesis, the peptide has the following covalent structure: HOOCCLYGKCRRYOGCSSASCCQR* (O = 4-trans hydroxyproline; * indicates an amidated C-terminus). The disulfide connectivity of the toxin is unrelated to the alpha- or the alphaA-conotoxins, the Conus peptide families that are competitive inhibitors of the nAChR, but shows homology to the mu-conotoxins (which are Na+ channel blockers).     

Cathepsin E expression by normal and premalignant cervical epithelium

We have investigated the expression of the aspartic proteinase cathepsin E and HLA-DR and the presence of HPV16 in normal squamous epithelium (n = 8) and low-grade (n = 21) and high-grade (n = 14) intraepithelial squamous lesions of the uterine cervix. Immunohistochemistry of cervical biopsies revealed that up-regulation of cathepsin E expression was related to increasing severity of the cervical intraepithelial neoplasia (CIN). Up-regulation of protein was associated with increased message as assessed by in situ hybridization. Langerhans cells and the majority of koilocytes did not express detectable cathepsin E levels. Although there was also an up-regulation of HLA-DR expression by cervical keratinocytes in cervical intraepithelial neoplasia lesions, as determined by immunohistochemistry, no significant correlation was found between HLA-DR and cathepsin E expression in these lesions; neither was expression of cathepsin E correlated to the presence of HPV16, detected by polymerase chain reaction. The expression of cathepsin E, an aspartic proteinase that is reported to play a role in antigen processing for presentation by class II major histocompatibility complex molecules, is associated with cellular dedifferentiation in cervical intraepithelial neoplasia.     

Cure of malignant ascites and generation of protective immunity by monoclonal antibody-targeted activation of a glucuronide prodrug in rats

We examined the in vivo efficacy of targeting beta-glucuronidase (betaG) to activate a glucuronide prodrug (BHAMG) of p-hydroxyaniline mustard (pHAM) at hepatoma ascites in Sprague-Dawley rats. Injection i.p. of 500 microg RH1-betaG, a conjugate formed between recombinant betaG and monoclonal antibody RH1 with specificity for an antigen expressed on AS-30D rat hepatoma cells, into rats bearing AS-30D ascites resulted in the accumulation of 54 microg conjugate per 10(9) tumor cells after 2 hr. Ascites fluid and serum contained 0.53 and 0 microg/ml, respectively, RH1-betaG 2 hr after injection of the conjugate. Conjugate binding to AS-30D cells was heterogeneous and non-saturated, as determined by flow cytometry. BHAMG was less toxic than pHAM to SD rats based on measures of animal mortality, weight loss and hematological toxicity. Treatment of rats bearing established hepatoma ascites with 500 microg RH1-betaG followed 2 hr later with a single i.p. injection of 30 mg/kg BHAMG or 3 i.p. injections of 10 mg/kg BHAMG 2, 3 and 4 hr later resulted in the cure of 6/8 and 8/8 animals, respectively. Treatment with BHAMG or pHAM alone did not produce cures, whereas treatment with a control antibody-betaG conjugate and BHAMG produced significantly greater hematological toxicity compared to treatment with RH1-betaG and BHAMG. All cured rats were completely protected from rechallenge with 2 x 10(7) AS-30D cells, indicating that successful treatment of animals induced protective immunity.     

Phosphoinositide 3-kinase induces scattering and tubulogenesis in epithelial cells through a novel pathway

Hepatocyte growth factor/scatter factor (HGF/SF) treatment of the Madin-Darby canine kidney epithelial cell line causes scattering of cells grown in monolayer culture and the formation of branching tubules by cells grown in collagen gels. HGF/SF causes prolonged activation of both the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase 2 (ERK2) and the phosphoinositide 3-OH kinase (PI 3-kinase) target protein kinase B (PKB)/Akt; inhibition of either the MAP kinase pathway by the MAP kinase/ERK kinase inhibitor PD98059 or the PI 3-kinase pathway by LY294002 blocks HGF/SF induction of scattering, although in morphologically distinct ways. Expression of constitutively activated PI 3-kinase, Ras, or R-Ras will cause scattering, but activated Raf will not, indicating that activation of the MAP kinase pathway is not sufficient for this response. Downstream of PI 3-kinase, activated PKB/Akt and Rac are both unable to induce scattering, implicating a novel pathway. Scattering induced by Ras or PI 3-kinase is sensitive to PD98059, as well as to LY294002, suggesting that basal MAP kinase activity is required, but not sufficient, for the scattering response. Induction of MDCK cell tubulogenesis in collagen gels by HGF/SF is inhibited by PD98059; expression of activated Ras and Raf causes disorganized growth in this system, but activated PI 3-kinase or R-Ras causes branching tubule formation similar to that seen with HGF/SF treatment. These data indicate that multiple signaling pathways acting downstream of Met and Ras are needed for these morphological effects; scattering is induced primarily by the PI 3-kinase pathway, which acts through effectors other than PKB/Akt or Rac and requires at least basal MAP kinase function. Elevated PI 3-kinase activity induces tubulogenesis, but total inhibition and excess activation of the MAP kinase pathway both oppose this effect.     

Stochastic model for a wavelike exothermal reaction in condensed heterogeneous systems

The labile protons of two 32-base-pair, four-arm models of immobile Holliday junctions have been studied by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. Overlap of resonances in the imino-imino region of two-dimensional nuclear Overhauser enhancement (NOE) spectra necessitates the use of a multi-pathway approach for obtaining sequence-specific assignments wherein all possible NOE connectivities to the labile protons are utilized, including those from the 2H of adenine, 5CH3 of thymine, and 5H of cytosine. Resonance assignments are obtained for all slowly exchanging imino and cytosine amino protons. Base-pairing up to and including the junction point is found in all four arms of both Holliday junctions. Several cross-arm NOE connectivities are identified and can be used to infer the geometry of the helical stacking domains. The two Holliday junctions studied, which differ only by the exchange of two base pairs at the branch point, appear to have opposite arm stacking geometries. These assignments form an important part of the critical background for detailed NMR analysis of Holliday junction structure and dynamics.