Trace metal contamination initiates the apparent auto-aggregation,amyloidosis, and oligomerization of Alzheimer’s Aβ peptides

Nucleation-dependent protein aggregation (seeding) and amyloid fibril-free formation of soluble SDS-resistant oligomers (oligomerization) by hydrophobic interaction is an in vitro model thought to propagate -amyloid (A) deposition, accumulation, and incur neurotoxicity and synaptotoxicity in Alzheimers disease (AD), and other amyloid-associated neurodegenerative diseases. However, A is a high-affinity metalloprotein that aggregates in the presence of biometals (zinc, copper, and iron), and neocortical A deposition is abolished by genetic ablation of synaptic zinc in transgenic mice. We now present in vitro evidence that trace (0.8 µM) levels of zinc, copper, and iron, present as common contaminants of laboratory buffers and culture media, are the actual initiators of the classic A1–42-mediated seeding process and A oligomerization. Replicating the experimental conditions of earlier workers, we found that the in vitro precipitation and amyloidosis of A1–40 (20 µM) initiated by A1–42 (2 µM) were abolished by chelation of trace metal contaminants. Further, metal chelation attenuated formation of soluble A oligomers from a cell-free culture medium. These data suggest that protein self-assembly and oligomerization are not spontaneous in this system as previously thought, and that there may be an obligatory role for metal ions in initiating A amyloidosis and oligomerization.     

Novel non-peptidic neuropeptide Y Y2 receptor antagonists

Through SAR studies of a piperidinylindoline cinnamide HTS lead, the first potent, non-peptide, low molecular weight selective Neuropeptide Y Y2 (NPY Y2) antagonists have been synthesized. The SAR studies around the piperidinyl, the indolinyl, and the cinnamyl moieties are discussed.     

Novel substituted 4-phenyl-[1,3]dioxanes: potent and selective orexin receptor 2 (OX(2)R) antagonists

Orexins, also termed hypocretins, consist of two neuropeptide agonists (orexin A and B) interacting with two known G-protein coupled receptors (OX(1)R and OX(2)R). In addition to other biological functions, the orexin-2 receptor is thought to be an important modulator of sleep and wakefulness. Herein we describe a series of novel, selective OX(2)R antagonists consisting of substituted 4-phenyl-[1,3]dioxanes. One such antagonist is compound 9, 1-(2,4-dibromo-phenyl)-3-((4S,5S)-2,2-dimethyl-4-phenyl-[1,3]dioxan-5-yl)-urea, which is bound by the OX(2)R with a pK(i) of 8.3, has a pK(b) of 7.9, and is 600-fold selective for the OX(2)R over the OX(1)R.     

The isolated N-terminal domain of the glucagon-like peptide-1 (GLP-1) receptor binds exendin peptides with much higher affinity than GLP-1

Two fragments of the receptor for glucagon-like peptide-1 (GLP-1), each containing the N-terminal domain, were expressed and characterized in either bacterial or mammalian cells. The first fragment, rNT-TM1, included the N-terminal domain and first transmembrane helix and was stably expressed in the membrane of human embryonic kidney 293 cells. The second, 6H-rNT, consisted of only the N-terminal domain of the receptor fused with a polyhistidine tag at its N terminus. The latter fragment was expressed in Escherichia coli in the form of inclusion bodies from which the protein was subsequently purified and refolded in vitro. Although both receptor fragments displayed negligible (125)I-labeled GLP-1(7-36)amide-specific binding, they both displayed high affinity for the radiolabeled peptide antagonist (125)I-exendin-4(9-39). Competition binding studies demonstrated that the N-terminal domain of the GLP-1 receptor maintains high affinity for the agonist exendin-4 as well as the antagonists exendin-4(3-39) and exendin-4(9-39) whereas, in contrast, GLP-1 affinity was greatly reduced. This study shows that although the exendin antagonists are not dependent upon the extracellular loops and transmembrane helices for maintaining their normal high affinity binding, the endogenous agonist GLP-1 requires regions outside of the N-terminal domain. Hence, distinct structural features in exendin-4, between residues 9 and 39, provide additional affinity for the N-terminal domain of the receptor. These data are consistent with a model for the binding of peptide ligands to the GLP-1 receptor in which the central and C-terminal regions of the peptides bind to the N terminus of the receptor, whereas the N-terminal residues of peptide agonists interact with the extracellular loops and transmembrane helices.     

Crystal structure of molybdopterin synthase and its evolutionary relationship to ubiquitin activation

Molybdenum cofactor (Moco) biosynthesis is an evolutionarily conserved pathway present in eubacteria, archaea and eukaryotes, including humans. Genetic deficiencies of enzymes involved in Moco biosynthesis in humans lead to a severe and usually fatal disease. Moco contains a tricyclic pyranopterin, termed molybdopterin (MPT), that bears the cis-dithiolene group responsible for molybdenum ligation. The dithiolene group of MPT is generated by MPT synthase, which consists of a large and small subunits. The 1.45 A resolution crystal structure of MPT synthase reveals a heterotetrameric protein in which the C-terminus of each small subunit is inserted into a large subunit to form the active site. In the activated form of the enzyme this C-terminus is present as a thiocarboxylate. In the structure of a covalent complex of MPT synthase, an isopeptide bond is present between the C-terminus of the small subunit and a Lys side chain in the large subunit. The strong structural similarity between the small subunit of MPT synthase and ubiquitin provides evidence for the evolutionary antecedence of the Moco biosynthetic pathway to the ubiquitin dependent protein degradation pathway.     

Cosecretion of chaperones and low-molecular-size medium additives increases the yield of recombinant disulfide-bridged proteins

Attempts were made to engineer the periplasm of Escherichia coli to an expression compartment of heterologous proteins in their native conformation. As a first approach the low-molecular-size additive L-arginine and the redox compound glutathione (GSH) were added to the culture medium. Addition of 0.4 M L-arginine and 5 mM reduced GSH increased the yield of a native tissue-type plasminogen activator variant (rPA), consisting of the kringle-2 and the protease domain, and a single-chain antibody fragment (scFv) up to 10- and 37-fold, respectively. A variety of other medium additives also had positive effects on the yield of rPA. In a second set of experiments, the effects of cosecreted ATP-independent molecular chaperones on the yields of native therapeutic proteins were investigated. At optimized conditions, cosecretion of E. coli DnaJ or murine Hsp25 increased the yield of native rPA by a factor of 170 and 125, respectively. Cosecretion of DnaJ also dramatically increased the amount of a second model protein, native proinsulin, in the periplasm. The results of this study are anticipated to initiate a series of new approaches to increase the yields of native, disulfide-bridged, recombinant proteins in the periplasm of E. coli.     

Do neocortical pyramidal neurons display stochastic resonance?

Neocortical pyramidal neurons in vivo are subject to an intense synaptic background activity that has a significant impact on various electrophysiological properties and dendritic integration. Using detailed biophysical models of a morphologically reconstructed neocortical pyramidal neuron, in which synaptic background activity was simulated according to recent measurements in cat parietal cortex in vivo, we show that the responsiveness of the cell to additional periodic subthreshold stimuli can be significantly enhanced through mechanisms similar to stochastic resonance. We compare several paradigms leading to stochastic resonance-like behavior, such as varying the strength or the correlation in the background activity. A new type of resonance-like behavior was obtained when the correlation was varied, in which case the responsiveness is sensitive to the statistics rather than the strength of the noise. We suggest that this type of resonance may be relevant to information processing in the cerebral cortex.     

Cardiac function and metabolism in Type 2 diabetic mice after treatment with BM 17.0744, a novel PPAR-alpha activator

Hearts from diabetic db/db mice, a model of Type 2 diabetes, exhibit left ventricular failure and altered metabolism of exogenous substrates. Peroxisome proliferator-activated receptor-alpha (PPAR-alpha) ligands reduce plasma lipid and glucose concentrations and improve insulin sensitivity in db/db mice. Consequently, the effect of 4- to 5-wk treatment of db/db mice with a novel PPAR-alpha ligand (BM 17.0744; 25-38 mg x kg(-1) x day(-1)), commencing at 8 wk of age, on ex vivo cardiac function and metabolism was determined. Elevated plasma concentrations of glucose, fatty acids, and triacylglycerol (34.0 +/- 3.6, 2.0 +/- 0.4, and 0.9 +/- 0.1 mM, respectively) were reduced to normal after treatment with BM 17.0744 (10.8 +/- 0.6, 1.1 +/- 0.1, and 0.6 +/- 0.1 mM). Plasma insulin was also reduced significantly in treated compared with untreated db/db mice. Chronic treatment of db/db mice with the PPAR-alpha agonist resulted in a 50% reduction in rates of fatty acid oxidation, with a concomitant increase in glycolysis (1.7-fold) and glucose oxidation (2.3- fold). Correction of the diabetes-induced abnormalities in systemic and cardiac metabolism after BM 17.0744 treatment did not, however, improve left ventricular contractile function.     

Diagnosis of a vulvar granular cell tumor by fine needle aspiration biopsy. A case report

BACKGROUND: Granular cell tumor (GCT) (granular cell "myoblastoma") is an uncommon neoplasm that may mimic carcinoma both clinically and morphologically. Fine needle aspiration diagnosis of vulvar GNT has been described on only one prior occasion. CASE: A 74-year-old, black female presented with a mass in the left labia. Fine needle aspiration biopsy revealed rare intact cells; abundant, granular, cytoplasmic fragments; and bland, ovoid, stripped nuclei. The intact cells were arranged in loose aggregates. Each sampling was exquisitely painful to the patient despite the use of local anesthesia. CONCLUSION: Cytologists should be aware of the distinctive clinical and morphologic appearance of GCT. The cytologic findings of vulvar, GCT are identical to those described at other body sites. Definitive diagnosis before extirpation permits definitive therapy.