Toxicity of a serotonin-derived neuromelanin

Postoperative Cognitive Dysfunction (POCD) is associated with increased mortality in the elderly and may occur from lipid peroxidation in aging. We previously showed that sevoflurane sequesters acrolein, which promotes the formation of a novel species of a putative neuromelanin. The current study examined the properties of this serotonin-derived melanoid (SDM). The interaction of SDM with unilamellar vesicles (ULVs) was examined using lipid membrane probes. Vesicle disruption was investigated by leakage of dye from calcein-loaded ULVs. We observed that SDM decreased diphenyl-hexatriene fluorescence anisotropy and increased the temperature-dependent change in anisotropy. SDM changed the absorbance of merocyanin-bound ULVs. SDM increased detergent-mediated calcein leakage. SDM structure was dramatically altered upon interaction with ULVs. We also observed that SDM enhanced detergent-mediated leakage of loaded ULVs, suggesting that SDM may be neurotoxic. We propose that inhalational agents, which sequester acrolein, may promote the production of certain species of neuromelanin that depletes local serotonin and enhances neuronal vulnerability.     

Interaction of human substantia nigra neuromelanin with lipids and peptides

Neuromelanin was isolated from human substantia nigra using different procedures. In the pigment isolated by any of these procedures a peptide component covalently bound to the melanic structure was found, as shown by treatment with reagents known to eliminate noncovalently bound proteins. The amino acid content of such a peptide component was reproducible and corresponded to approximately 15% of the neuromelanin weight. Neuromelanin also showed the ability to absorb specifically lipid molecules, approximately 20% of its weight, and among these lipids cholesterol was identified, constituting approximately 5% of the total lipid mixture. A synthetic melanin, incubated with putamen homogenate, bound tissue peptides with an amino acid content quite close to that of neuromelanin. The same synthetic melanin adsorbed a lower amount of lipids from the putamen homogenate compared with neuromelanin. The sulfur content of neuromelanin was also reproducible even using different isolation procedures. A nonpigmented tissue like corpus callosum was used as a control and extracted by the method used for neuromelanin isolation; a total elimination of tissue components was found, thus demonstrating the capability of the reported procedures to isolate neuromelanin alone. The presence of a peptide component in the neuromelanin structure and the selective affinity for lipid molecules suggest new aspects of the functional role and metabolic pathway of neuromelanin.     

The absolute concentration of nigral neuromelanin, assayed by a new sensitive method, increases throughout the life and is dramatically decreased in Parkinson’s disease

The concentration of neuromelanin (NM) in substantia nigra pars compacta (SNPC) has been measured in male and female normal subjects at different ages in the range 1–97 years old and in SNPC of parkinsonian patients. A very similar age trend of NM concentration was found in both sexes. In the first year of life NM was not detectable, between 10 and 20 years the NM levels were 0.3–0.8 μg/mg of SNPC, between 20 and 50 years were 0.8–2.3 μg/mg SNPC and between 50 and 90 were 2.3–3.7 μg/mg of SNPC. In parkinsonian subjects, the NM levels were 1.2–1.5 μg/mg of SNPC, which is less than 50% with respect to the age-matched controls. These data demonstrate a continuous NM accumulation in SNPC neurons during aging, the presence of large amounts of NM in SNPC and severe depletion of NM in Parkinson’s disease.     

Structure of the human serotonin 5-HT4 receptor gene and cloning of a novel 5-HT4 splice variant

Several variants of the serotonin 5-HT4 receptor are known to be produced by alternative splicing. To survey the existence and usage of exons in humans, we cloned the human 5-HT4 gene. Based on sequence analysis seven C-terminal variants (a-g) and one internal splice variant (h) were found. We concentrated in this study on the functional characterization of the novel splice variant h, which leads to the insertion of 14 amino acids into the second extracellular loop of the receptor. The h variant was cloned as a splice combination with the C-terminal b variant; therefore, we call this receptor 5-HT4(hb). This novel receptor variant was expressed transiently in COS-7 cells, and its pharmacological profile was compared with those of the previously cloned 5-HT4(a) and 5-HT4(b) isoforms, with the latter being the primary reference for the h variant. In competition binding experiments using reference 5-HT4 ligands, no significant differences were detected. However, the broadly used 5-HT4 antagonist GR113808 discriminated functionally among the receptor variants investigated. As expected, it was an antagonist on the 5-HT4(a) and 5-HT4(b) variant but showed partial agonistic activity on the 5-HT4(hb) variant. These data emphasize the importance of variations introduced by splicing for receptor pharmacology and may help in the understanding of conflicting results seen with 5-HT4 ligands in different model systems.     

NMR reveals a novel glutaredoxin-glutaredoxin interaction interface

Glutaredoxins (Grx) represent a large family of glutathione (GSH)-dependent oxidoreductases that catalyse the reduction of disulfides or glutathione mixed disulfide. Grx domains from pathogenic bacteria and plant Grxs have been recently reported to target specific peroxiredoxins (Prxs). The specificity that triggers the interaction between Grx and Prx is poorly understood and is only based on the structure of Haemophilus influenzae Prx-Grx hybrid (hyPrx5). We report here an NMR study of the Populus tremula Grx C4 that targets a P.tremula D-type II Prx. We show that Grx C4 specifically self-associates in a monomer-dimer equilibrium with an apparent K(d) of ca 2.6 mM. Grx C4 homodimer was docked under experimental restraints. The results reveal a novel Grx-Grx interface that is unrelated to the hyPrx5 Grx-Grx dimer interface. Chemical-shift perturbations and 15N spin-relaxation measurements show that the auto-association surface comprises both the active site and the GSH binding site. Reduced GSH is demonstrated to bind reduced Grx with a K(d) of ca 8.6 mM. The potential biological significance of the new Grx-Grx interaction interface is discussed.     

Ultrastructure of neuromelanin granules in dog substantia nigra at different ages

The Authors study through ultrastructural observations the sequence of processes concerning the appearance and the depositing of the neuromelanic pigment in the neurons of dog mesencephalic substantia nigra nucleus at different ages. The results are as follows: in the one year old dog, in a low percentage of lysosome-like oval organelles, few masses, strongly electron-dense, appear on the matrix. In the five year old dog, these masses are deposited and increased in quantity inside many more organelles. In the specimens extracted from older dogs, these organelles, characterized moreover by the presence of lipidic globules, merging in groups, became larger and more irregular. The Authors discuss the results obtained with the ultrastructural observation, compared with those obtained with the optical methods.     

The neuromelanin of the human substantia nigra

The pigment of the human substantia nigra was isolated after extraction of lipids and proteins with 2% sodium cholate in 30% ethanol followed by 2% sodium dodecyl sulfate in 10% glycerol. The pigment was hydrolysed with HI or degraded by treatment with KMNO4 and the samples were examined for compounds known to derive from pheomelanin (4-amino-3-hydroxyphenylalanine, AHP and 4-amino-3-hydroxyphenylethylamine, AHPEA), or from eumelanin (pyrrole-2,3,5-tricarboxylic acid, PTCA). The HI hydrolysis yielded AHPEA in large quantities, indicating cysteinyldopamine as the main source of the pheomelanin moiety of the neuromelanin, but also trace amounts of AHP, derived from cysteinyldopa oxidation products. Dopamine and small quantities of dopa were also obtained by HI hydrolysis of the neuromelanin. The yield of PTCA was low, but the amounts observed show that part of the neuromelanin is of the eumelanin type, a fact compatible with an occasional exhaustion of the glutathione-cysteine reduction system at the site of neuromelanin formation.     

Phosphorylation at the interface

Proteomic studies have identified thousands of eukaryotic phosphorylation sites (phosphosites), but few are functionally characterized. Nishi et?al., in this issue of Structure, characterize phosphosites at protein-protein interfaces and estimate the effect of their phosphorylation on interaction affinity, by combining proteomics data with protein structures.     

Abeta-polyacrolein aggregates: novel mechanism of plastic formation in senile plaques

High levels of acrolein (H2C=HC-CH=O) occur in Alzheimer's brain. Amyloid-beta (Abeta) peptide co-localizes with acrolein presumably due to Abeta-induced lipid peroxidation. Focal production of acrolein may yield a transient elevation in the concentration of acrolein that may be susceptible to polymerization via basic latex polymer chemistry. Following incubation of Abeta with acrolein (16-750 mM), we observed the formation of thin plastic fragments that were extensively punctuated. Planar aggregates stained for protein and for cross-beta structures suggesting an Abeta-polyacrolein colloidal mixture. Depending on acrolein concentration and incubation time, we observed uniformly sized planar aggregates (approximately 10 microm2) or monolayers (>100 mm2) of thin polyacrolein films embedded with Abeta oligomers. The ability of Abeta to catalyze the polymerization of acrolein is likely due to Abeta's surfactant and redox properties. These observations suggest that plastic in the form of Abeta-polyacrolein latexes may exist in neural tissue contributing to the pathogenesis of Alzheimer's disease.     

Phytochemicals at the plant-insect interface

Opportunities for genetic engineering of natural products are increasing, while discovery and development of synthetic insecticides and developmental regulators are declining. However, discovery and potential applications of natural compounds are constrained by present ecological knowledge and theory. Biochemistry offers additional perspective to chemical interaction across the interface between plant and herbivore. Phytochemical effects on an insect herbivore may be determined by physical, chemical, and biotic characteristics of the microenvironment during phytochemical transfer between plant and insect. The midgut lumen is often overlooked as part of this microenvironment. It initially determines rates of metabolism and uptake of phytochemicals into hemolymph, and ultimately the quantity of a compound seen by affected tissues. Additive processes such as absorption, binding, and transport by proteins in hemolymph may ultimately prove more crucial to toxication than subtractive processes such as metabolism and excretion. Uptake and transport of coumarins in hemolymph are being studied in larvae of the citrus root weevil Diaprepes abbreviatus. Studies with synthetic 7-amino-3-phenyl coumarin (coumarin-10) have preceded studies with natural coumarins. The fluorescence properties of coumarin-10 have enabled determination of absorption and binding to hemolymph proteins.     

Adhesion of Leptospira at a solid-liquid interface: a model

Two strains of the saprophytic Leptospira biflexa serovar patoc display reversible and irreversible adhesion at a solid-liquid interface. Both forms of adhesion are enhanced in the presence of 20 M carbonyl cyanide metachlorophenyl hydrazone (CCCP), an uncoupler which inhibits motility of the bacteria. Microscopic observations also indicated that motility may have a role in adhesion as only actively motile organisms were seen to detach from the substratum. A dynamic model is proposed for adhesion of these organisms at a solid-liquid interface. It is suggested that the level of reversible adhesion is determined by the comparative rates of attachment (ON phase) and detachment (OFF phase). As reversible adhesion is mediated by weak forces of attraction, bacterial motility or gentle washing could promote the OFF phase. When motility is inhibited, the OFF phase is reduced and the ON phase continues (as motility is not required for the ON phase) causing the level of reversible adhesion to increase. Since reversible adhesion is a prerequisite for irreversible adhesion, then increased reversible adhesion leads directly to increased irreversible adhesion. Reversible adhesion appears to be mediated by the weak attractive forces of the secondary minimum whereas the mechanism facilitating irreversible adhesion of leptospires is not known.Abbreviation CCCP carbonyl cyanide meta-chlorophenyl hydrazone     

Neuromelanins in various regions of human brain are associated with native and oxidized isoprenoid lipids

Neuromelanin (NM) isolated from seven regions of the human brain is found to contain series of natural and oxidized isoprenoid lipids. Specifically, dolichols (dol) and dolichoic acids (dol-CA) with 14-22 and 14-21 isoprene units are identified. Standards of nor-dolichol and nor-dolichoic acid were used to determine the relative amounts of dol and dol-CA compared to the total lipids present in NM for each region. The cerebellum, putamen, globus pallidus, and premotor cortex contained similar amounts of dol, comprising approximately 8-9.5% of the total lipid weight. Interestingly, the corpus callosum contains substantially lower quantities of both dol and dol-CA compared to the other regions—less than 4% dol relative to the total lipid weight. Oxidized and reduced dolichol-related species were identified and determined to be region-dependent.     

Interactions at the bilayer interface and receptor site induced by the novel synthetic pyrrolidinone analog MMK3

This work presents a thorough investigation of the interaction of the novel synthetic pyrrolidinone analog MMK3 with the model membrane system of dipalmitoylphosphatidylcholine (DPPC) and the receptor active site. MMK3 has been designed to exert antihypertensive activity by functioning as an antagonist of the angiotensin II receptor of subtype 1 (AT₁). Its low energy conformers were characterized by 2D rotating-frame Overhauser effect spectroscopy (ROESY) in combination with molecular dynamics (MD) simulations. Docking study of MMK3 shows that it fits to the AT₁ receptor as SARTANs, however, its biological activity appears to be lower. Thus, differential scanning calorimetry (DSC), Raman spectroscopy and small angle X-ray scattering (SAXS) experiments on the interaction of MMK3 with DPPC bilayers were carried out and results demonstrate that the drug is well incorporated into the membrane leaflets and furthermore causes partial bilayer interdigitation, although less effective than SARTANs. Thus, it appears that the nature of the bilayer matrix and the stereoelectronic active site requirements of the receptor are responsible for the low bioactivity of MMK3.     

Keratinocytes grown at the air-liquid interface

Summary A procedure is described which allows primary cultures of rat keratinocytes grown at the liquid-air interface to develop and maintain multilayered strata and to produce highly keratinized sheets morphologically similar to those seen in epidermis in situ. Various substrata were tested and compared as to their ability to support growth and stratification of keratinocytes. It was found that when cultured on plastic surfaces, keratinocytes adhered tightly to the substratum and produced a confluent monolayer that later stratified to two to three layers. Cells plated on Vitrogen 100 collagen failed to reach confluence and, in addition, exhibited the “clustering” phenomenon and deterioration of collagen after 3 to 4 d of growth. Significantly better attachment and spreading were observed for cells grown on rat-tail collagen as compared with plastic and Vitrogen 100 collagen. The best results, including maximal and uniform stratification, were seen in cells grown on a mixture, of rat-tail and Vitrogen 100 collagens. The system that was developed in the present study offers a model for use in the study of epidermal toxicity from topically applied environmental chemicals. This investigation was supported by grant 5 T32 AM 07236 and 5 R01 AM 15206 from the National Institutes of Health, contract DAMD17-82-C-2198 from the US Army Medical Research and Development Command, and contract N-2 from the Proctor and Gamble Company The contents of this paper do not represent the opinion of the sponsors nor should findings in this report be construed as their official position.