Intracellular calcium buffering capacity in isolated squid axons

Changes in ionized calcium were studied in axons isolated from living squid by measuring absorbance of the Ca binding dye Arsenazo III using multiwavelength differential absorption spectroscopy. Absorption changes measured in situ were calibrated in vitro with media of ionic composition similar to axoplasm containing CaEGTA buffers. Calcium loads of 50-2,500 μmol/kg axoplasm were induced by microinjection, by stimulation in 112 mM Ca seawater, or by soaking in choline saline with 1-10 mM Ca. Over this range of calcium loading of intact axoplasm, the ionized calcium in the axoplasm rose about 0.6 nM/μM load. Similar loading in axons preteated with carbonyl cyanide 4- trifluoromethoxyphenylhydrazone (FCCP) to inhibit the mitochondrial proton gradient increased ionized calcium by 5-7 percent of the imposed load, i.e. 93-95 percent of the calcium load was buffered by a process insensitive to FCCP. This FCCP- insensitive buffer system was not saturated by the largest calcium loads imposed, indicating a capacity of at least several millimolar. Treatment of previously loaded axons with FCCP or apyrase plus cyanide produced rises in ionized calcium which could be correlated with the extent of the load. Analysis of results indicated that, whereas only 6 percent of the endogenous calcium in fresh axons is stored in the FCCP-sensitive (presumably mitochondrial) buffer system, about 30 percent of an imposed exogenous load in the range of 50-2,500 μM is taken up by this system.     

3D modeling and characterization of the human CD115 monoclonal antibody H27K15 epitope and design of a chimeric CD115 target

The humanized monoclonal antibody H27K15 specifically targets human CD115, a type III tyrosine kinase receptor involved in multiple cancers and inflammatory diseases. Binding of H27K15 to hCD115 expressing cells inhibits the functional effect of colony-stimulating factor-1 (CSF-1), in a non-competitive manner. Both homology modeling and docking programs were used here to model the human CD115 extracellular domains, the H27K15 variable region and their interaction. The resulting predicted H27K15 epitope includes mainly the D1 domain in the N-terminal extracellular region of CD115 and some residues of the D2 domain. Sequence alignment with the non-binding murine CD115, enzyme-linked immunosorbent assay, nuclear magnetic resonance spectroscopy and affinity measurements by quartz crystal microbalance revealed critical residues of this epitope that are essential for H27K15 binding. A combination of computational simulations and biochemical experiments led to the design of a chimeric CD115 carrying the human epitope of H27K15 in a murine CD115 backbone that is able to bind both H27K15 as well as the murine ligands CSF-1 and IL-34. These results provide new possibilities to minutely study the functional effects of H27K15 in a transgenic mouse that would express this chimeric molecule.     

Microbial Community Comparisons as a Function of the Physical and Geochemical Conditions of Galápagos Island Fumaroles

Microbial communities at six fumarole fields on Sierra Negra and Alcedo volcanoes in the Galápagos Islands were examined to test how extreme geochemical conditions affect microbial biodiversity. The geologic substrates consist of basalt and rhyolite with varying amounts of alteration and sulfur precipitates. Collected samples of substrates varied in pH from 0–6, and substrate temperatures were within the mesophilic, thermophilic, and hyperthermophilic temperature ranges. Terminal restriction fragment length polymorphism (T-RFLP) analyses were done to assess the relationship of communities to each other as a function of geologic substrate, pH, and temperature. Comparative analyses of community diversity define two distinct clusters showing that the relationship between spatially separated microbial communities at the fumaroles is most influenced by the pH of the local environment.     

Lipocalin Q83 reveals a dual ligand binding mode with potential implications for the functions of siderocalins

Siderocalins are particular lipocalins that participate in the innate immune response by interfering with bacterial siderophore-mediated iron uptake. Additionally, siderocalins are involved in several physiological and pathological processes such as inflammation, iron delivery, tissue differentiation, and cancer progression. Here we show that siderocalin Q83 displays an unexpected dual ligand binding mode as it can bind enterobactin and unsaturated fatty acids simultaneously. The solution structure of the siderocalin Q83 in complex with arachidonic acid and enterobactin reveals molecular details of this novel dual binding mode and the determinants of fatty acid binding specificity. Our results suggest that Q83 is a metabolic hub linking iron and fatty acid pathways. This unexpected coupling might contribute to the pleiotropic functions of siderocalins.     

Thalamo-Basal Ganglia Connectivity in Postmenopausal Women Receiving Estrogen Therapy

Cumulative data on the effects of estrogen therapy (ET) on brain function in postmenopausal women suggests that ET influences cerebral metabolism and may protect against age-related declines in various domains of cognitive function. The beneficial cognitive effects of ET may relate to its modulation of the thalamic-striatum cholinergic and dopaminergic systems, as the activity of both neurotransmitter systems in the thalamus appears to be positively influenced by estrogen. In the current study, we attempted to evaluated regional cerebral brain metabolism utilizing [18F]-fluorodeoxyglucose positron emission tomography in 11 healthy recently-postmenopausal women on ET (ET+) in comparison to 11 recently-postmenopausal and ET-naïve women (ET?) in order to assess the effects of ET on cholinergic and dopaminergic system regulation. Results showed thalamo-basal ganglia connectivity among ET+ women but not among ET? women. The presence of connectivity in the thalamo-striatal pathway in recently postmenopausal women suggests estrogen effects in preserving integrity of the cholinergic and dopaminergic systems. The results also suggest that ET initiated at or near the menopausal transition may modulate brain aging by mediating complex sensory-motor functions.     

A specific inhibitor of cysteine proteases impairs a Vif-dependent modification of human immunodeficiency virus type 1 Env protein.

The Vif protein of human immunodeficiency virus type 1 (HIV-1) regulates viral infectivity. Virions produced in cell culture after transfection by a Vif-negative molecular clone show a dramatic decrease in infectivity for susceptible CD4+ cell lines, although the Vif protein does not appear to be a constituent of the viral particle. The exact mechanism by which Vif affects HIV-1 infectivity is so far unknown. We report the existence of structural homologies between Vif and a family of cysteine proteases and present evidence which suggests that one of the targets of Vif is the Env protein and more precisely the cytoplasmic domain of gp41. Vif was found to modify both the processing and conformation of the Env protein. Ethyl(25, 35)- 3[(5)-3-methyl-1-(3-methylbutylcarbamoyl)]oxirane-2-carboxylate, a specific inhibitor of cysteine proteases, inhibits the effect of Vif, as does the mutation of Cys-114 to Leu in Vif. Furthermore, Cys-114 of Vif produced in Escherichia coli, interacts directly with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane. These observations suggest that a cysteine protease activity is associated with Vif and that this activity plays a role in Env maturation.