Haemoglobin components and oxygen transport in relation to habitat distribution in triplefin fishes (Tripterygiidae)

Haemoglobin components were analysed for nine species of New Zealand triplefins and their isoelectric points (pI) ranged from 5.1 to 7.0. The number of well-expressed isohaemoglobins was larger in shallow-water and tidal pool species, ranging from four in Grahamina signata to eight in Grahamina capito, and were relatively cathodal. Two strongly anodal isohaemoglobins were expressed in the mid-depth species Ruanoho decemdigitatus and Ruanoho whero, and one in the deeper water species Karalepis stewarti and Forsterygion malcolmi. The red blood cell oxygen-binding properties were determined at 15 °C and 25 °C in the pH range 6.7–7.9 for the shallow-water species G. capito, the shallow to mid-depth species Forsterygion varium, and the deep-water species F. malcolmi. Oxygen affinity was highest for G. capito and the magnitude of the Bohr effect lower (Δlog P ₅₀/ΔpH = −0.37 at 25 °C, where P ₅₀ is the half-saturation coefficient) compared to the two Forsterygion species (Δlog P ₅₀/ΔpH = −0.52 to −0.59). Further, the cooperativity factor, n ₅₀, was lower in G. capito thus maintaining oxygen transport over a wide range of environmental oxygen pressures. Oxygen binding was similarly influenced by temperature in both G. capito and F. malcolmi (maximum heat of oxygenation ΔH_max = −27 kJ mol⁻¹ and −37 kJ mol⁻¹, respectively). Thus, triplefin fishes living in shallow, thermally unstable habitats possess a greater number of cathodally migrating isohaemoglobins, and their red blood cells have a higher oxygen affinity and reduced cooperativity which is less sensitive to changes in pH than do species occurring in more stable, deeper water habitats. Our analysis of an assemblage of closely related species circumvents some of the difficulties inherent in studies where interpretation of experimental results is confounded by phylogeny. Accepted: 18 March 1999     

Epidermal growth factor (EGF)-like repeats of human tenascin-C as ligands for EGF receptor

Signaling through growth factor receptors controls such diverse cell functions as proliferation, migration, and differentiation. A critical question has been how the activation of these receptors is regulated. Most, if not all, of the known ligands for these receptors are soluble factors. However, as matrix components are highly tissue-specific and change during development and pathology, it has been suggested that select growth factor receptors might be stimulated by binding to matrix components. Herein, we describe a new class of ligand for the epidermal growth factor (EGF) receptor (EGFR) found within the EGF-like repeats of tenascin-C, an antiadhesive matrix component present during organogenesis, development, and wound repair. Select EGF-like repeats of tenascin-C elicited mitogenesis and EGFR autophosphorylation in an EGFR-dependent manner. Micromolar concentrations of EGF-like repeats induced EGFR autophosphorylation and activated extracellular signal-regulated, mitogen-activated protein kinase to levels comparable to those induced by subsaturating levels of known EGFR ligands. EGFR-dependent adhesion was noted when the ligands were tethered to inert beads, simulating the physiologically relevant presentation of tenascin-C as hexabrachion, and suggesting an increase in avidity similar to that seen for integrin ligands upon surface binding. Specific binding to EGFR was further established by immunofluorescence detection of EGF-like repeats bound to cells and cross-linking of EGFR with the repeats. Both of these interactions were abolished upon competition by EGF and enhanced by dimerization of the EGF-like repeat. Such low affinity behavior would be expected for a matrix-"tethered" ligand; i.e., a ligand which acts from the matrix, presented continuously to cell surface EGF receptors, because it can neither diffuse away nor be internalized and degraded. These data identify a new class of "insoluble" growth factor ligands and a novel mode of activation for growth factor receptors.     

Receptor tyrosine kinase ERBB4 mediates acquired resistance to ERBB2 inhibitors in breast cancer cells

Approximately 25% of breast cancers overexpress and depend on the receptor tyrosine kinase ERBB2, one of 4 ERBB family members. Targeted therapies directed against ERBB2 have been developed and used clinically, but many patients continue to develop resistance to such therapies. Although much effort has been focused on elucidating the mechanisms of acquired resistance to ERBB2-targeted therapies, the involvement of ERBB4 remains elusive and controversial. We demonstrate that genetic ablation of ERBB4, but not ERBB1-3, led to apoptosis in lapatinib-resistant cells, suggesting that the efficacy of pan-ERBB inhibitors was, at least in part, mediated by the inhibition of ERBB4. Moreover, ERBB4 was upregulated at the protein level in ERBB2+ breast cancer cell lines selected for acquired lapatinib resistance in vitro and in MMTV-Neu mice following prolonged lapatinib treatment. Knockdown of ERBB4 caused a decrease in AKT phosphorylation in resistant cells but not in sensitive cells, suggesting that ERBB4 activated the PI3K/AKT pathway in lapatinib-resistant cells. Importantly, ERBB4 knockdown triggered apoptosis not only in lapatinib-resistant cells but also in trastuzumab-resistant cells. Our results suggest that although ERBB4 is dispensable for naïve ERBB2+ breast cancer cells, it may play a key role in the survival of ERBB2+ cancer cells after they develop resistance to ERBB2 inhibitors, lapatinib and trastuzumab.     

Single cocaine exposure does not alter striatal pre‐synaptic dopamine function in mice: an [18F]‐FDOPA PET study

Cocaine is a recreational drug of abuse that binds to the dopamine transporter, preventing reuptake of dopamine into pre‐synaptic terminals. The increased presence of synaptic dopamine results in stimulation of both pre‐ and post‐synaptic dopamine receptors, considered an important mechanism by which cocaine elicits its reinforcing properties. However, the effects of acute cocaine administration on pre‐synaptic dopamine function remain unclear. Non‐invasive imaging techniques such as positron emission tomography have revealed impaired pre‐synaptic dopamine function in chronic cocaine users. Similar impairments have been seen in animal studies, with microdialysis experiments indicating decreased basal dopamine release. Here we use micro positron emission tomography imaging techniques in mice to measure dopamine synthesis capacity and determine the effect of acute cocaine administration of pre‐synaptic dopamine function. We show that a dose of 20 mg/kg cocaine is sufficient to elicit hyperlocomotor activity, peaking 15–20 min post treatment (p < 0.001). However, dopamine synthesis capacity in the striatum was not significantly altered by acute cocaine treatment (: 0.0097 per min vs. 0.0112 per min in vehicle controls, p > 0.05). Furthermore, expression levels of two key enzymes related to dopamine synthesis, tyrosine hydroxylase and aromatic l ‐amino acid decarboxylase, within the striatum of scanned mice were not significantly affected by acute cocaine pre‐treatment (p > 0.05). Our findings suggest that while the regulation of dopamine synthesis and release in the striatum have been shown to change with chronic cocaine use, leading to a reduced basal tone, these adaptations to pre‐synaptic dopaminergic neurons are not initiated following a single exposure to the drug.

     

Determining the effects of life stage,shared prey density and host plant on intraguild predation of a native lacewing (<Emphasis Type="Italic">Chrysoperla carnea</Emphasis>) by an invasive coccinellid (<Emphasis Type="Italic">Harmonia axyridis</Emphasis>)

Negative impacts of non-native Harmonia axyridis (Pallas) on members of the native aphid enemy guild have been widely hypothesised but mainly only assessed with other coccinellid species, and mostly in small experimental arenas. Here we investigated the interactions between H. axyridis and Chrysoperla carnea Stephens larvae. In small-scale (Petri dish) arenas 2nd-instar C. carnea were at risk of predation from larval (2nd and 4th-instar) and adult (male and female) H. axyridis while 3rd-instar C. carnea were only at minimal risk from 4th-instar and adult female H. axyridis. Plant species, aphid species and aphid density did not affect intraguild predation of 2nd-instar C. carnea by 4th-instar and adult H. axyridis in mesocosm experiments. Chrysoperla carnea consumed similar numbers of Megoura viciae Buckton, Aphis fabae Scop. and Acyrthosiphon pisum Harris aphids while H. axyridis consumed fewer M. viciae than the other two species. The greatest suppression of A. pisum was achieved in treatments with both C. carnea and H. axyridis. Life stage and the sex of H. axyridis as well as the life stage of C. carnea are important variables affecting intraguild predation and these attributes should be considered when assessing the potential threat of other potentially invasive alien predators.     

A structural and dynamic model for the interaction of interleukin-8 and glycosaminoglycans: support from isothermal fluorescence titrations

Binding of interleukin-8 (IL-8) to glycosaminoglycans (GAGs) on the surface of endothelial cells is crucial for the recruitment of neutrophils to an inflammatory site. Deriving structural knowledge about this interaction from in silico docking experiments has proved difficult because of the high flexibility and the size of GAGs. Therefore, we developed a docking method that takes into account ligand and protein flexibility by running approximately 15,000 molecular dynamics simulations of the docking event with different initial orientations of the binding partners. The method was shown to successfully reproduce the residues of basic fibroblast growth factor involved in GAG binding. Docking of a heparin hexasaccharide to IL-8 gave an interaction interface involving the basic residues His18, Lys20, Arg60, Lys64, Lys67, and Arg68. By subjecting IL-8 single-site mutants, in which these amino acids were replaced by alanine, to isothermal fluorescence titrations, the affinities for heparin were determined to be wtIL-8 > IL-8(H18A) > IL-8(R68A) > IL-8(K67A) > IL-8(K20A) > IL-8(R60A) > IL-8(K64A). A comparison with the binding energies calculated from the model revealed high values for wtIL-8 and the H18A mutant and significantly lower but similar energies for the remaining mutants. Connecting the two fully sulfated hexasaccharides bound to each of the two IL-8 monomers in the dimeric chemokine by an N-acetylated dodecasaccharide gave a complex structure in which the GAG molecule aligned in a parallel fashion to the N-terminal alpha-helices of IL-8 like a horseshoe. A 5-ns molecular dynamics simulation of this complex confirmed its structural stability and revealed a reorientation in both binding sites where a disaccharide became the central binding unit. Isothermal fluorescence titration experiments using differently sulfated heparin disaccharides confirmed that a single disaccharide can indeed bind IL-8 with high affinity.     

Isolation of a clone containing human histone genes.

A recombinant clone containing human histone genes has been isolated. The clone, lambda HH-01, was selected from a genomal library using chicken histone cDNA and a cloned fragment containing chicken histone genes as probes. Sub-clones from lambda HH-01 have been mapped and coding regions located with cDNA. The human H3 gene has been identified by DNA sequence analysis.     

p115 Rho GTPase activating protein interacts with MEKK1

Mammalian MAP/ERK kinase kinase 1 (MEKK1) was identified as a mammalian homolog of Ste11p of the yeast pheromone-induced mating pathway. Like Ste11p, MEKK1 is a MAP3 kinase linked to at least two MAP kinase cascades and regulatory events that require cytoskeletal reorganization. MEKK1 is activated by molecules that impact cytoskeletal function. MEKK1-/-cells are defective in cell migration, demonstrating that it is required for cell motility. MEKK1 has a 1,200 residue N-terminal regulatory domain that interacts with a dozen identified proteins. Using part of the MEKK1 N-terminus in a yeast two-hybrid screen, we discovered a novel interaction with p115 Rho GTPase-activating protein (GAP). The p115 Rho GAP binds to MEKK1 in vitro and in intact cells. The p115 Rho GAP has selectivity for RhoA over other Rho family members. Expression of p115 Rho GAP reduces MEKK1-induced signaling to AP-1. The reduced activation of AP-1 is dependent on the association of MEKK1 with p115 Rho GAP, because deletion of the Rho GAP SH3 domain, which abrogates their interaction, restores the stimulatory effect of MEKK1 on AP-1 activity. Here we have identified an MEKK1 binding partner that offers a connection between this protein kinase and the machinery regulating cytoskeletal reorganization.