Likely year-round presence of beaked whales in the Bay of Biscay

Hydrobiologia - Beaked whales are cryptic and difficult to study species, often distributed in deep offshore waters and only briefly visible at the surface. A diverse range of cetacean species has...     

Dasatinib Modulates Invasive and Migratory Properties of Canine Osteosarcoma and has Therapeutic Potential in Affected Dogs

BACKGROUND: This investigation sought to elucidate the relationship between hepatocyte growth factor (HGF)–induced metastatic behavior and the tyrosine kinase inhibitors (TKIs) crizotinib and dasatinib in canine osteosarcoma (OS). Preliminary evidence of an apparent clinical benefit from adjuvant therapy with dasatinib in four dogs is described. METHODS: The inhibitors were assessed for their ability to block phosphorylation of MET; reduce HGF-induced production of matrix metalloproteinase (MMP); and prevent invasion, migration, and cell viability in canine OS cell lines. Oral dasatinib (0.75 mg/kg) was tested as an adjuvant therapy in four dogs with OS. RESULTS: Constitutive phosphorylation of MET was detected in two cell lines, and this was unaffected by 20-nM incubation with either dasatinib or crizotinib. Incubation of cell lines with HGF (MET ligand) increased cell migration and invasion in both cell lines and increased MMP-9 activity in one. Dasatinib suppressed OS cell viability and HGF-induced invasion and migration, whereas crizotinib reduced migration and MMP-9 production but did not inhibit invasion or viability. CONCLUSIONS: Invasion, migration, and viability of canine OS cell lines are increased by exogenous HGF. HGF induces secretion of different forms of MMP in different cell lines. The HGF-driven increase in viability and metastatic behaviors we observed are more uniformly inhibited by dasatinib. These observations suggest a potential clinical benefit of adjuvant dasatinib treatment for dogs with OS.     

Quenching of singlet oxygen by alkaloids and related nitrogen heterocycles

Fifteen plant alkaloids and related heterocyclic compounds were tested for their ability to quench singlet oxygen. Most of the compounds showed high activity; brucine and strychnine were especially efficient quenchers. Brucine, at a concentration of ca 2.6 x 10^?5 M, is capable of inactivating half the singlet oxygen molecules it encounters. This quenching may serve in nature to protect plants from the deleterious effects of singlet oxygen or other reactive oxidants.     

Comparison of immunoaffinity columns with Florisil/C18 columns for the determination of aflatoxins in animal feed and maize

A study of the performance of the EC method and an Immunoaffinity method for aflatoxins B1,B2,G1 and G2 in animal feed and maize was performed. The extraction and HPLC analysis of both procedures were comparable,hence a direct comparison of the performance of the alternative clean- up columns was made. Throughout the study the results were similar for both methods,however,the main advantage of the Immunoaffinity method was fewer manipulations were required. Therefore the method was easier to use,less solvent was required and greater sample throughput (approximately 2 fold) was obtained.     

Long-term primary culture of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver

Summary Long-term primary cultures of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver have been established. Nearly homogenous (>97%) populations of hepatocytes were placed into primary culture and remained viable and proliferative for at least 70 d. In addition to hepatocytes, proliferative biliary cells persisted in the cultures for at least 30 d. Finally, a third type of epithelial cell, which we have termed a “spindle cell,” consistently appeared and proliferated to confluence in these cultures. The confluent cultures of spindle cells were successfully subcultured and passaged. The initial behavior, growth, and optimization of serum and media requirements for these cells is described. All three cell types proliferated as measured by thymidine incorporation, autoradiography, proliferating cellular nuclear antigen analysis, and propidium iodine staining. Further efforts to characterize the cells included western blotting and immunohistochemical staining with antibodies to cytokeratins previously reported in fish liver. From these data, it appears that all three cell populations are epithelial in nature. Furthermore, significant changes in actin organization, often indicative of transformation or pluripotent cells, were observed with increased time in primary culture.     

Effects of Opioid Peptides Containing the Sequence of Met5-Enkephalin or Leu5-Enkephalin on Nicotine-Induced Secretion from Bovine Adrenal Chromaffin Cells

Eighteen endogenous opioid peptides, all containing the sequence of either Met5- or Leu5-enkephalin, were tested for their ability to modify nicotine-induced secretion from bovine adrenal chromaffin cells. ATP released from suspensions of freshly isolated cells was measured with the luciferin-luciferase bioluminescence method as an index of secretion. None of the peptides affected 5 microM nicotine-induced ATP release at 10 nM. Three peptides inhibited secretion at 5 microM: dynorphin1-13, dynorphin1-9, and rimorphin inhibited by 65%, 37%, and 29% respectively. Use of peptidase inhibitors (bestatin, thiorphan, bacitracin, or 1,10-phenanthroline) did not result in any of the other peptides showing potent actions on the nicotinic response, although bestatin and thiorphan did enhance the inhibitory actions of dynorphin1-13 and dynorphin1-9 by 20-30%. Nicotine-induced secretion of endogenous catecholamines from bovine chromaffin cells cultured for 3 days was also studied to assess any selective actions of the peptides on adrenaline or noradrenaline cell types. Dynorphin1-13 was 1,000-fold more potent than Leu5-enkephalin at inhibiting endogenous catecholamine secretion. Dynorphin1-13 was slightly more potent at inhibiting noradrenaline release than adrenaline release whereas Leu5-enkephalin showed the opposite selectivity. The structure-activity relationships of opioid peptide actions on the chromaffin cell nicotinic response are discussed in relation to the properties of the adrenal opioid binding sites.     

Further characterization of benzodiazepine receptor differences in long-sleep and short-sleep mice

Molecular and conformational characteristics of benzodiazepine (BZ) receptors in cortex and cerebellum from long-sleep and short-sleep mice were investigated using heat inactivation and beta-carboline competition techniques. To investigate differences in the allosteric coupling between GABA and BZ receptors, the protection of BZ receptors from heat inactivation, by GABA, was also evaluated. The two genotypes do not differ in the affinity or number of BZ receptors in the cortex or cerebellum. They do, however, appear to differ in the molecular structure and/or regulation of the conformational state of the receptor in the cortex, as indicated by a greater sensitivity of LS mice to both heat inactivation and beta-carboline competition of 3H-flunitrazepam (FNZ) binding in this region. Evidence for differences in the nature of coupling between GABA and BZ receptors is provided by the finding that in both regions, GABA protected BZ receptors from inactivation to a greater degree in LS mice. The relationship between these differences and the multiplicity of expression of BZ receptors is discussed.     

Extraction Techniques for Gastrins and Cholecystokinins in the Rat Central Nervous System

Abstract: Samples of rat striatum and synthetic sul-phated cholecystokinin octapeptide were extracted by different procedures and the solubilised cholecystokinin-like immunoreactivity analysed by gel filtration and ion-exchange chromatography. Ice-cold 90% methanol extraction gave the greatest recovery of tissue immunoreactivity without any major modification of the extracted components. The 33-amino acid form of cholecystokinin was poorly recovered by this extractant. Boiling water or a combined boiling water/acetic acid extraction gave efficient recovery of tissue immunoreactivity but chemically modified a substantial part of the octapeptide-like component. Boiling in acetic acid alone also produced this modification but in addition resulted in poor recovery of the octapeptide-like component. The combined water/ acetic acid extraction gave reasonable to good recovery of all added Cholecystokinins and gastrins, including the 33-amino acid form of cholecystokinin. Ice-cold 0.1 M HCl was less efficient than 90% methanol at solubilising tissue immunoreactivity and resulted in a substantial modification of the octapeptide component distinct from that produced by boiling extractions, possibly desulpha-tion. The results show that more than one extraction procedure is needed to study all the cholecystokinin components in brain tissue and demonstrates the necessity of using at least two chromatographic systems for such studies.     

Distribution and Chromatographic Characterisation of Gastrin and Cholecystokinin in the Rat Central Nervous System

Abstract: Two tissue extraction techniques and two radioimmunoassays were used to study the distribution of gastrin and cholecystokinin in rat brain. Small amounts of gastrin were found in extracts of neurohypophysis, but in neither ice-cold 90% methanol nor in boiling water-acetic acid extracts of the other 33 brain areas studied. Cholecystokinin was found in equivalent amounts in both types of extract of 31 areas. The distribution was similar to that in previous studies. The components of cholecystokinin immunoreactivity were characterised in 10 rat CNS tissues using four tissue extraction methods in conjunction with gel filtration and ion-exchange chromatography. The results demonstrated that gastrins were present only in the neurohypophysis and that in all other rat CNS tissues the main molecular component was indistinguishable from the sulphated octapeptide of cholecystokinin. Minor immunoreactive components were observed in all types of extract of all tissues with the properties of the desulphated octapeptide and the C-terminal tetrapeptide amide, suggesting they are genuine tissue components, not extraction artefacts. Large molecular forms of cholecystokinin were not detected in any tissue. The results emphasise the necessity of using two or more extraction methods and two or more chromatography systems in such a study.     

Development of an assay for histamine using automated high-performance liquid chromatography with electrochemical detection

Previous studies have measured histamine by derivatization with o-phthaldialdehyde (OPA) and mercaptoethanol (ME), followed by reversed-phase HPLC separation and electrochemical detection. The derivatization product, however, was very unstable. In the present study, inclusion of less polar solvents (e.g., acetonitrile or tetrahydrofuran) in the OPA/ME derivatization reaction produced an OPA/ME-histamine product that was stable for many hours. Changes of the HPLC mobile phase (increasing its ionic strength and pH and including triethylamine) dramatically improved the chromatography and reduced the histamine detection limit to <0.1 pmol. The modified assay was suitable for batchwise manual derivatization of histamine samples followed by their automated analysis by HPLC with an automatic injector.