New melanic pigments in the human brain that accumulate in aging and block environmental toxic metals.

Neuronal pigments of melanic type were identified in the putamen, cortex, cerebellum, and other major regions of human brain. These pigments consist of granules 30 nm in size, contained in organelles together with lipid droplets, and they accumulate in aging, reaching concentrations as high as 1.5-2.6 microg/mg tissue in major brain regions. These pigments, which we term neuromelanins, contain melanic, lipid, and peptide components. The melanic component is aromatic in structure, contains a stable free radical, and is synthesized from the precursor molecule cysteinyl-3,4-dihydroxyphenylalanine. This contrasts with neuromelanin of the substantia nigra, where the melanic precursor is cysteinyl-dopamine. These neuronal pigments have some structural similarities to the melanin found in skin. The precursors of lipid components of the neuromelanins are the polyunsaturated lipids present in the surrounding organelles. The synthesis of neuromelanins in the various regions of the human brain is an important protective process because the melanic component is generated through the removal of reactive/toxic quinones that would otherwise cause neurotoxicity. Furthermore, the resulting melanic component serves an additional protective role through its ability to chelate and accumulate metals, including environmentally toxic metals such as mercury and lead.

Neuromelanins isolated from different regions of the human brain exhibit a common surface photoionization threshold.

Neuromelanin isolated from the premotor cortex, cerebellum, putamen, globus pallidus and corpus callosum of the human brain is studied by scanning probe and photoelectron emission microscopies and the results are compared with previously published work on neuromelanin from the substantia nigra. Scanning electron microscopy reveals common structure for all neuromelanins. All exhibit spherical entities of diameters between 200 and 400 nm, composed of smaller spherical substructures, approximately 30 nm in diameter. These features are similar to that observed for many melanin systems including Sepia cuttlefish, bovine eye, and human eye and hair melanosomes. Photoelectron microscopy images were collected for all neuromelanins at specific wavelengths of ultraviolet light between 248 and 413 nm, using the spontaneous emission output from the Duke free electron laser. Analysis of the data establishes a common threshold photoionization potential for neuromelanins of 4.7 +/- 0.2 eV, corresponding to an oxidation potential of -0.3 +/- 0.2 V vs the normal hydrogen electrode (NHE). These results are consistent with previously reported potentials for neuromelanin from the substantia nigra of 4.5 +/- 0.2 eV (-0.1 +/- 0.2 V vs NHE). All neuromelanins exhibit a common low surface oxidation potential, reflecting their eumelanic component and their inability to trigger redox processes with neurotoxic effect.

Determining thermodynamic parameters from isothermal calorimetric isotherms of the binding of macromolecules to metal cations originally chelated by a weak ligand.

An accurate data analysis method for determining stoichiometry and thermodynamic parameters from isothermal titration calorimetry data for the binding of macromolecules to metal cations that are solubilized through an association with a weak ligand is presented. This approach is applied to determine the binding constant for the association of Cu(II) to the first 16 residues of the Alzheimer's amyloid beta peptide, Abeta(1-16) under conditions where Cu(II) is rendered soluble through weak binding to glycine. At pH 7.2 and 37 degrees C, a binding constant of 1.5 x 10(9) M-1 (Kd = 0.7 nM) is determined for the association of Cu(II) with Abeta(1-16).

Quantification of the binding constant of copper(II) to the amyloid-beta peptide.

The amyloid beta (A beta) peptide of Alzheimer's disease binds copper(II), and the peptide-bound metal may be a source of reactive oxygen species and neurotoxicity. To circumvent peptide aggregation and reduce redox activity, there is growing interest in using metal chelates as drug therapeutics for AD, whose design requires accurate data on the affinity of A beta peptides for copper(II). Reports on Cu2+ binding to A beta range from approximately 10(5) to approximately 10(9); these values' being obtained for different peptide lengths (1-16, 1-28, 1-40, 1-42) at varying pH. Herein, we report that Cu2+'s binding to A beta(1-40) at 37 degrees C occurs in a 1:1 stoichiometry with a pH-dependent binding constant: 1.1 (+/-0.2) x 10 (9) M (-1) and 2.4 (+/-0.2) x 10 (9) M(-1) at pH 7.2 and 7.4, respectively. Under identical conditions, A beta(1-16) reveals a comparable binding constant, confirming that this portion of the peptide is the binding region. Several previously reported values can be reconciled with the current measurement by careful consideration of thermodynamics associated with the presence of competing ligands used to solubilize copper.

Robotic-assisted resection of isolated paraaortic lymph node recurrence with right lateral decubitus position.

In this report we describe transperitoneal robotic-assisted paraaortic lymphadenectomy via the right lateral decubitus position to treat solitary recurrence in a patient with cervical carcinoma. This is, to our knowledge, the first report utilizing the right lateral decubitus position rather than the traditional approach with the Trendelenberg position. This approach adds another option for surgical approaches to the paraaortic lymph nodes, particularly in subgroups of patients who have significant cardiopulmonary cormobidities and are unable to tolerate the steep Trendelenburg position.

Quantification of the binding properties of Cu2+ to the amyloid beta peptide: coordination spheres for human and rat peptides and implication on Cu2+-induced aggregation.

There is no consensus on the coordinating ligands for Cu(2+) by Abeta. However, the differences in peptide sequence between human and rat have been hypothesized to alter metal ion binding in a manner that alters Cu(2+)-induced aggregation of Abeta. Herein, we employ isothermal titration calorimetry (ITC), circular dichroism (CD), and electron paramagnetic resonance (EPR) spectroscopy to examine the Cu(2+) coordination spheres to human and rat Abeta and an extensive set of Abeta(16) mutants. EPR of the mutant peptides is consistent with a 3N1O binding geometry, like the native human peptide at pH 7.4. The thermodynamic data reveal an equilibrium between three coordination spheres, {NH(2), O, N(Im)(His6), N(-)}, {NH(2), O, N(Im)(His6), N(Im)(His13)}, and {NH(2), O, N(Im)(His6), N(Im)(His14)}, for human Abeta(16) but one dominant coordination for rat Abeta(16), {NH(2), O, N(Im)(His6), N(-)}, at pH 7.4-6.5. ITC and CD data establish that the mutation R5G is sufficient for reproducing this difference in Cu(2+) binding properties at pH 7.4. The substitution of bulky and positively charged Arg by Gly is proposed to stabilize the coordination {NH(2), O-, N(Im)(His6), N(-)} that then results in one dominating coordination sphere for the case of the rat peptide. The differences in the coordination geometries for Cu(2+) by the human and rat Abeta are proposed to contribute to the variation in the ability of Cu(2+) to induce aggregation of Abeta peptides.

Quantification of Ca(2+) binding to melanin supports the hypothesis that melanosomes serve a functional role in regulating calcium homeostasis.

Calcium regulation in melanocytes affects numerous biological pathways including protecting the redox balance in the cell and regulating the supply of substrate, l-tyrosine, for melanogenesis. The pigment contained in the melanocytes, melanin, has been implicated in maintaining calcium homeostasis in the cell and is known to be involved with calcium ion regulation in the inner ear. Herein, the association constant for Ca(2+) binding to Sepia melanin is determined by isothermal titration calorimetry to be 3.3 (+/-0.2) x 10(3)/M. This value is comparable with other well-established intracellular calcium-binding proteins that serve to buffer calcium concentrations, lending further support to the hypothesis that melanosomes serve as intracellular mediators of calcium homeostasis in melanocytes. Using this binding constant and the data from a fluorescent Ca(2+) displacement assay, the pK(a) of the carboxyl group coordinated to Ca(2+) is determined to be 3.1 +/- 0.1.

The surface oxidation potential of human neuromelanin reveals a spherical architecture with a pheomelanin core and a eumelanin surface.

Neuromelanin (NM) isolated from the substantia nigra region of the human brain was studied by scanning probe and photoelectron emission microscopies. Atomic force microscopy reveals that NM granules are comprised of spherical structures with a diameter of approximately 30 nm, similar to that observed for Sepia cuttlefish, bovine eye, and human eye and hair melanosomes. Photoelectron microscopy images were collected at specific wavelengths of UV light between 248 and 413 nm, using the spontaneous-emission output from the Duke OK-4 free electron laser. Analysis of the data establishes a threshold photoionization potential for NM of 4.5 +/- 0.2 eV, which corresponds to an oxidation potential of -0.1 +/- 0.2 V vs. the normal hydrogen electrode (NHE). The oxidation potential of NM is within experimental error of the oxidation potential measured for human eumelanosomes (-0.2 +/- 0.2 V vs. NHE), despite the presence of a significant fraction of the red pigment, pheomelanin, which is characterized by a higher oxidation potential (+0.5 +/- 0.2 V vs. NHE). Published kinetic studies on the early chemical steps of melanogenesis show that in the case of pigments containing a mixture of pheomelanin and eumelanin, of which NM is an example, pheomelanin formation occurs first with eumelanin formation predominantly occurring only after cysteine levels are depleted. Such a kinetic model would predict a structural motif with pheomelanin at the core and eumelanin at the surface, which is consistent with the measured surface oxidation potential of the approximately 30-nm constituents of NM granules.