4. ONE YEAR'S CENSUS AND STUDY OF BIRDS IN 2½ ACRES OF ALEANY BUSHVELD. (Part 2.) April, 1945, to March, 1946

Hooded Vultures Necrosyrtes monachus are critically endangered but little is known of their year-round use of nests or whether other species usurp Hooded Vulture nest sites. We investigated visitation rates by Hooded Vultures and other species (including potential nest predators and usurpers) to examine their effect on Hooded Vulture breeding success. We present observations of 33 species recorded by camera traps at 12 Hooded Vulture nests over a total of 93 nest-months (2 095 nest-days). Several pairs of Hooded Vultures visited their nests regularly during the non-breeding season, some adding nesting material, highlighting that pairs visited their nest(s) year round. Egyptian Geese Alopochen aegyptiaca, potential usurpers of raptor nests, were present at occupied and unoccupied Hooded Vulture nests, but we recorded no usurpation of nests by Egyptian Geese and they had no impact on vulture breeding success. Hooded Vulture breeding failure was linked to two species only: camera-trap imagery recorded one case of predation of a vulture egg by a Chacma Baboon Papio ursinus, and one case of a Martial Eagle Polemaetus bellicosus predating a vulture nestling. We recommend expanding the Hooded Vulture nest monitoring programme to include more pairs.     

On the diet of the Pharaoh Eagle Owl,Bubo ascalaphus (Savigny, 1809), in Qatar,with an overview of its feeding habits

The diet of Bubo ascalaphus in Qatar was assessed based on pellets collected from the first known nesting site of the species in the country. The pellets contained a total of 68 prey items, representing 9 different species: 4 mammals, 1 bird, 1 reptile, and at least 3 scorpions. Mammals clearly comprised the major food source (89.7% and 97.7% in frequency and biomass respectively). Our data suggest that Pharaoh Eagle Owls are opportunistic predators that feed on a variety of prey depending on their temporal/spatial availability, which is consistent with previous studies. A literature review clearly suggests that Eagle Owls in arid to semi-arid environments are opportunistic predators with small mammals being their main prey. Predation on migrating Blue-cheeked Bee-eaters Merops persicus supports this hypothesis.     

Mitigation of radiation injury by selective stimulation of the LPA2 receptor

Due to its antiapoptotic action, derivatives of the lipid mediator lysophosphatidic acid (LPA) provide potential therapeutic utility in diseases associated with programmed cell death. Apoptosis is one of the major pathophysiological processes elicited by radiation injury to the organism. Consequently, therapeutic explorations applying compounds that mimic the antiapoptotic action of LPA have begun. Here we present a brief account of our decade-long drug discovery effort aimed at developing LPA mimics with a special focus on specific agonists of the LPA₂ receptor subtype, which was found to be highly effective in protecting cells from apoptosis. We describe new evidence that 2-((3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)thio)benzoic acid (GRI977143), a prototypic nonlipid agonist specific to the LPA₂ receptor subtype, rescues apoptotically condemned cells in vitro and in vivo from injury caused by high-dose γ-irradiation. GRI977143 shows the features of a radiomitigator because it is effective in rescuing the lives of mice from deadly levels of radiation when administered 24 h after radiation exposure. Our findings suggest that by specifically activating LPA₂ receptors GRI977143 activates the ERK1/2 prosurvival pathway, effectively reduces Bax translocation to the mitochondrion, attenuates the activation of initiator and effector caspases, reduces DNA fragmentation, and inhibits PARP-1 cleavage associated with γ-irradiation-induced apoptosis. GRI977143 also inhibits bystander apoptosis elicited by soluble proapoptotic mediators produced by irradiated cells. Thus, GRI977143 can serve as a prototype scaffold for lead optimization paving the way to more potent analogs amenable for therapeutic exploration. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Desmin mutations in the terminal consensus motif prevent synemin-desmin heteropolymer filament assembly

Disorganization of the desmin network is associated with cardiac and skeletal myopathies characterized by accumulation of desmin-containing aggregates in the cells. Multiple associations of intermediate filament proteins form a network to increase mechanical and functional stability. Synemin is a desmin-associated type VI intermediate filament protein. Neither its impact on desmin network nor how it integrates into desmin filament is yet elucidated. To gain more insight into the molecular basis of these processes, we coexpressed synemin with different desmin mutants in ex vivo models. The screening of fourteen desmin mutants showed that synemin with desmin mutants revealed two behaviors. Firstly, synemin was co-localized in desmin aggregates and its coexpression decreased the number of cells containing aggregates. Secondly, synemin was excluded from the aggregates, then synemin had no effect on desmin network organization. Among fourteen desmin mutants, there were only three mutants, p.E401K, p.R406W and p.E413K, in which synemin was not found in aggregates. This behavior was correlated to the abnormal salt-bridges of desmin-dimer as seen in silico constructs. Moreover, desmin constructs in silico and published results in literature have predicted that the salt-bridges absence in the desmin filament building prevent longitudinal annealing and/or radial compaction. These results suggest that the state of desmin-filament assembly is crucial for synemin anchorage and consequently might involve mechanical and functional stability of the cytoskeletal network.     

Calmyrin1 binds to SCG10 protein (stathmin2) to modulate neurite outgrowth

Calmyrin1 (CaMy1) is an EF-hand Ca²⁺-binding protein expressed in several cell types, including brain neurons. Using a yeast two-hybrid screen of a human fetal brain cDNA library, we identified SCG10 protein (stathmin2) as a CaMy1 partner. SCG10 is a microtubule-destabilizing factor involved in neuronal growth during brain development. We found increased mRNA and protein levels of CaMy1 during neuronal development, which paralleled the changes in SCG10 levels. In developing primary rat hippocampal neurons in culture, CaMy1 and SCG10 colocalized in cell soma, neurites, and growth cones. Pull-down, coimmunoprecipitation, and proximity ligation assays demonstrated that the interaction between CaMy1 and SCG10 is direct and Ca²⁺-dependent in vivo and requires the C-terminal domain of CaMy1 (residues 99-192) and the N-terminal domain of SCG10 (residues 1-35). CaMy1 did not interact with stathmin1, a protein that is homologous with SCG10 but lacks the N-terminal domain characteristic of SCG10. CaMy1 interfered with SCG10 inhibitory activity in a microtubule polymerization assay. Moreover, CaMy1 overexpression inhibited SCG10-mediated neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells. This CaMy1 activity did not occur when an N-terminally truncated SCG10 mutant unable to interact with CaMy1 was expressed. Altogether, these data suggest that CaMy1 via SCG10 couples Ca²⁺ signals with the dynamics of microtubules during neuronal outgrowth in the developing brain. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Dinitrosyliron complexes are the most abundant nitric oxide-derived cellular adduct: biological parameters of assembly and disappearance

It is well established that nitric oxide (^•NO) reacts with cellular iron and thiols to form dinitrosyliron complexes (DNIC). Little is known, however, regarding their formation and biological fate. Our quantitative measurements reveal that cellular concentrations of DNIC are proportionally the largest of all ^•NO-derived adducts (900 pmol/mg protein, or 45-90 μM). Using murine macrophages (RAW 264.7), we measured the amounts, and kinetics, of DNIC assembly and disappearance from endogenous and exogenous sources of ^•NO in relation to iron and O₂ concentration. Amounts of DNIC were equal to or greater than measured amounts of chelatable iron and depended on the dose and duration of ^•NO exposure. DNIC formation paralleled the upregulation of iNOS and occurred at low physiologic ^•NO concentrations (50-500 nM). Decreasing the O₂ concentration reduced the rate of enzymatic ^•NO synthesis without affecting the amount of DNIC formed. Temporal measurements revealed that DNIC disappeared in an oxygen-independent manner (t_1/2 = 80 min) and remained detectable long after the ^•NO source was removed (> 24 h). These results demonstrate that DNIC will be formed under all cellular settings of ^•NO production and that the contribution of DNIC to the multitude of observed effects of ^•NO must always be considered.     

Neuregulin-1β regulates outgrowth of neurites and migration of neurofilament 200 neurons from dorsal root ganglial explants in vitro

Neuregulin-1β (NRG-1β) signaling has multiple functions in neurons. To assess NRG-1β on neurite outgrowth and neuronal migration in vitro, organotypic dorsal root ganglion (DRG) neuronal culture model was established. Neurite outgrowth and neuronal migration were evaluated using this culture model in the presence (5 nmol/L, 10 nmol/L, 20 nmol/L) or absence of NRG-1β. Neurofilament 200 (NF-200)-immunoreactive (IR) neurons were determined as the migrating neurons. The number of nerve fiber bundles extended from DRG explant increased significantly in the presence of NRG-1β (5 nmol/L, 23.0 ± 2.2, P < 0.05; 10 nmol/L, 27.0 ± 2.7, P < 0.001; 20 nmol/L, 30.8 ± 3.7, P < 0.001) as compared with that in the absence of NRG-1β (19.0 ± 2.2). The number of neurons migrating from DRG explants increased significantly in the presence of NRG-1β (5 nmol/L, 39.6 ± 5.0, P < 0.05; 10 nmol/L, 54.6 ± 6.7, P < 0.001; 20 nmol/L, 62.2 ± 5.7, P < 0.001) as compared with that in the absence of NRG-1β (31.6 ± 4.0). Moreover, the increase of the number of nerve fiber bundles and the number of migrating NF-200-IR neurons was dose-dependent for NRG-1β addition. The data in this study imply that NRG-1β promotes neurite outgrowth and neuronal migration from DRG explants in vitro.     

Regulation of glutamate metabolism by hydrocortisone and branched chain keto acids in cultured rat retinal Müller cells (TR-MUL)

Glutamate released from retinal neurons during neurotransmission is taken up by retinal Müller cells, where much of the amino acid is subsequently amidated to glutamine or transaminated to α-ketoglutarate for oxidation. Müller cell glutamate levels may have to be carefully maintained at fairly low concentrations to avoid excesses of glutamate in extracellular spaces of the retina that would otherwise cause excitotoxicity. We employed a cultured rat retinal Müller cell line in order to study the metabolism and the role of Müller cell specific enzymes on the glutamate disposal pathways. We found that the TR-MUL cells express the glial specific enzymes, glutamine synthetase, the mitochondrial isoform of branched chain aminotransferase (BCATm) and pyruvate carboxylase, all of which are involved in glutamate metabolism and homeostasis in the retina. Hydrocortisone treatment of TR-MUL cells increased glutamine synthetase expression and the rate of glutamate amidation to glutamine. Addition of branched chain keto acids (BCKAs) increased lactate and aspartate formation from glutamate and also oxidation of glutamate to CO₂ and H₂O. The two glutamate disposal pathways (amidation and oxidation) did not influence each other. When glutamate levels were independently depleted within TR-MUL cells, the uptake of glutamate from the extracellular fluid increased compared to uptake from control (undepleted) cells suggesting that the level of intracellular glutamate may influence clearing of extracellular glutamate.     

Monoclonal antibody to serum immunoglobulins of Clarias batrachus and its application in immunoassays

Serum immunoglobulins of Clarias batrachus (Cb-Ig) were purified by affinity chromatography using bovine serum albumin as capture ligand. Under reducing conditions in SDS-PAGE, Cb-Ig was composed of a heavy (H) chain (68.7 kDa) and two light (L) chains (27.4 and 26.3 kDa). Purified Cb-Ig was used to produce a monoclonal antibody (MAb) designated E4 MAb that belonged to IgG1 subclass. In Western blotting, this MAb showed binding to H chain of purified Cb-Ig and putative H chains in reduced sera of C. batrachus, Clarias gariepinus and Heteropneustes fossilis. However, no binding was observed with serum protein of Labeo rohita and Channa striata. Cross-reactivity of anti-Cb-Ig MAb was observed with serum of C. batrachus, C. gariepinus and H. fossilis in competitive ELISA. In immunoblotting of non-reduced Cb-Ig with E4 MAb, four bands assumed to be tetrameric, trimeric, dimeric and monomeric form were observed. In flow cytometric analysis of the gated lymphocytes, the number of surface Ig-positive (Ig +) cells in blood, spleen, kidney and thymus of C. batrachus was determined to be 50.1 ± 3.1, 55.1 ± 3.36, 42.4 ± 4.81 and 5.1 ± 0.89%, respectively, using E4 MAb. Ig + cells were also demonstrated in formalin-fixed paraffin embedded tissue sections of spleen, kidney, thymus and smears of blood mononuclear cells in indirect immunoperoxidase test. The developed MAb was employed to detect pathogen-specific immunoglobulins in the sera of C. batrachus immunized with killed Edwardsiella tarda, by an indirect ELISA. This monoclonal antibody can be useful tool in immunological research and assays.