Quinine-induced agranulocytosis: toxic effect of quinine bisulphate on bone marrow cultures in vitro.

In a case of quinine-induced agranulocytosis marrow culture studies confirmed the inhibitory effect on the patient''s cells of equivalent therapeutic plasma concentrations of quinine. Similar concentrations had no effect on normal marrow cells. Quinidine, the stereoisomer of quinine, had no effect on either cells from the patient or normal cells. The results encourage the use of in-vitro bone marrow cultures for identifying drugs responsible for agranulocytosis.     

Neuropeptide Y (NPY) cleaving enzymes: structural and functional homologues of dipeptidyl peptidase 4

N-terminal truncation of NPY has important physiological consequences, because the truncated peptides lose their capability to activate the Y1-receptor. The sources of N-terminally truncated NPY and related peptides are unknown and several proline specific peptidases may be involved. First, we therefore provide an overview on the peptidases, belonging to structural and functional homologues of dipeptidyl peptidase 4 (DP4) as well as aminopeptidase P (APP) and thus, represent potential candidates of NPY cleavage in vivo. Second, applying selective inhibitors against DP4, DP8/9 and DP2, respectively, the enzymatic distribution was analyzed in brain extracts from wild type and DP4 deficient F344 rat substrains and human plasma samples in activity studies as well as by matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF)-mass spectrometry. Third, co-transfection of Cos-1 cells with Dpp4 and Npy followed by confocal lasermicroscopy illustrated that hNPY-dsRed1-N1 was transported in large dense core vesicles towards the membrane while rDP4-GFP-C1 was transported primarily in different vesicles thereby providing no clear evidence for co-localization of NPY and DP4. Nevertheless, the review and experimental results of activity and mass spectrometry studies support the notion that at least five peptidases (DP4, DP8, DP9, XPNPEP1, XPNPEP2) are potentially involved in NPY cleavage while the serine protease DP4 (CD26) could be the principal peptidase involved in the N-terminal truncation of NPY. However, DP8 and DP9 are also capable of cleaving NPY, whereas no cleavage could be demonstrated for DP2.     

Analyzing proteome topology and function by automated multidimensional fluorescence microscopy

Temporal and spatial regulation of proteins contributes to function. We describe a multidimensional microscopic robot technology for high-throughput protein colocalization studies that runs cycles of fluorescence tagging, imaging and bleaching in situ. This technology combines three advances: a fluorescence technique capable of mapping hundreds of different proteins in one tissue section or cell sample; a method selecting the most prominent combinatorial molecular patterns by representing the data as binary vectors; and a system for imaging the distribution of these protein clusters in a so-called toponome map. By analyzing many cell and tissue types, we show that this approach reveals rules of hierarchical protein network organization, in which the frequency distribution of different protein clusters obeys Zipf's law, and state-specific lead proteins appear to control protein network topology and function. The technology may facilitate the development of diagnostics and targeted therapies.     

N-Terminal pyroglutamate formation of Aβ38 and Aβ40 enforces oligomer formation and potency to disrupt hippocampal long-term potentiation

Pyroglutamate (pGlu)-modified amyloid peptides have been identified in sporadic and familial forms of Alzheimer's disease (AD) and the inherited disorders familial British and Danish Dementia (FBD and FDD). In this study, we characterized the aggregation of amyloid-β protein Aβ37, Aβ38, Aβ40, Aβ42 and ADan species in vitro, which were modified by N-terminal pGlu (pGlu-Aβ3-x, pGlu-ADan) or possess the intact N-terminus (Aβ1-x, ADan). The pGlu-modification confers rapid formation of oligomers and short fibrillar aggregates. In accordance with these observations, the pGlu-modified Aβ38, Αβ40 and Αβ42 species inhibit hippocampal long term potentiation of synaptic response, but pGlu-Aβ3-42 showing the highest effect. Among the unmodified Aβ peptides, only Aβ1-42 exhibites such propensity, which was similar to pGlu-Aβ3-38 and pGlu-Aβ3-40. Likewise, the amyloidogenic peptide pGlu-ADan impaired synaptic potentiation more pronounced than N-terminal unmodified ADan. The results were validated using conditioned media from cultivated HEK293 cells, which express APP variants favoring the formation of Aβ1-x, Aβ3-x or N-truncated pGlu-Aβ3-x species. Hence, we show that the ability of different amyloid peptides to impair synaptic function apparently correlates to their potential to form oligomers as a common mechanism. The pGlu-modification is apparently mediating a higher surface hydrophobicity, as shown by 1-anilinonaphtalene-8-sulfonate fluorescence, which enforces potential to interfere with neuronal physiology.     

Potassium-Stimulated Taurine Release and Nitric Oxide Synthase Activity During Quinolinic Acid Lesion of the Rat Striatum

The microdialysis technique was used to study the effect of nitric oxide synthase (NOS) activity on taurine release. Taurine release was characterized in rat striatum that was excitotoxically lesioned compared to normal conditions. The basal taurine level of the dialysate decreased during quinolinate (QUIN) lesion in parallel to the cell degeneration process. The K⁺-stimulated taurine concentration also decreased during QUIN-lesion, but to an extent that was different from that of basal values. K⁺-stimulated taurine levels were further markedly lowered by coapplication of the NOS inhibitor L-NAME in control and in lesioned animals up to 30 days after QUIN-injection. Postdegenerative tissue did not show any NOS-dependency in K⁺-induced taurine release. We conclude that a substantial part of K⁺-induced taurine release depends on NOS-activity both in normal brain tissue and in excitotoxically induced neurodegeneration. The main source of K⁺-induced taurine release in control rats are neurons but in lesioned animals are activated astroglial cells.     

Isoaspartate-containing amyloid precursor protein-derived peptides alter efficacy and specificity of potential beta-secretases

Neuritic plaques of Alzheimer patients are composed of multiple protein components. Among them, the amyloid beta-peptides (Abeta) 1-40/42 and further N- and C-terminally modified fragments of Abeta are highly abundant. Most prominent are the isoaspartate (isoAsp)-Abeta peptides and pyroglutamyl (pGlu)-Abeta. While pGlu-Abeta can only be formed from an N-terminal glutamate by glutaminyl cyclase, spontaneous isoAsp-isomerization cannot occur at an N-terminal aspartate of peptides. This means that isoAsp-Abeta formation must precede proteolysis of the amyloid precursor protein (APP). Abeta generation from APP by beta- and gamma-secretases initiates the amyloid peptide aggregation and deposition process. Two aspartate proteases have been identified as secretases: BACE-1 (beta-site amyloid precursor protein cleaving enzyme) and the intramembrane gamma-secretase multiprotein complex. However, recent evidence supports more than one beta-secretase initiating this cascade. Formation of Abeta1-40/42 was predominantly studied by expression of mutated human APP sequences in cell culture and transgenic animals, generating Abeta fragments that did not contain such multiple posttranslational modifications as in Alzheimer's disease. This prompted us to investigate the catalytic turnover of Asp- or isoAsp-containing APP-derived peptide sequences by BACE-1 and cathepsin B, another potential beta-secretase. While cathepsin B is more effective than BACE-1 in processing the Asp-containing peptide derivatives, only cathepsin B can cleave the isoAsp-containing peptides, which occurs with high catalytic efficiency.     

Complementarity of structure ensembles in protein-protein binding

Protein-protein association is often accompanied by changes in receptor and ligand structure. This interplay between protein flexibility and protein-protein recognition is currently the largest obstacle both to our understanding of and to the reliable prediction of protein complexes. We performed two sets of molecular dynamics simulations for the unbound receptor and ligand structures of 17 protein complexes and applied shape-driven rigid body docking to all combinations of representative snapshots. The crossdocking of structure ensembles increased the likelihood of finding near-native solutions. The free ensembles appeared to contain multiple complementary conformations. These were in general not related to the bound structure. We suggest that protein-protein binding follows a three-step mechanism of diffusion, free conformer selection, and refolding. This model combines previously conflicting ideas and is in better agreement with the current data on interaction forces, time scales, and kinetics.     

Exceptional Evolutionary Divergence of Human Muscle and Brain Metabolomes Parallels Human Cognitive and Physical Uniqueness

Metabolite concentrations reflect the physiological states of tissues and cells. However, the role of metabolic changes in species evolution is currently unknown. Here, we present a study of metabolome evolution conducted in three brain regions and two non-neural tissues from humans, chimpanzees, macaque monkeys, and mice based on over 10,000 hydrophilic compounds. While chimpanzee, macaque, and mouse metabolomes diverge following the genetic distances among species, we detect remarkable acceleration of metabolome evolution in human prefrontal cortex and skeletal muscle affecting neural and energy metabolism pathways. These metabolic changes could not be attributed to environmental conditions and were confirmed against the expression of their corresponding enzymes. We further conducted muscle strength tests in humans, chimpanzees, and macaques. The results suggest that, while humans are characterized by superior cognition, their muscular performance might be markedly inferior to that of chimpanzees and macaque monkeys.     

Selective inhibition of dipeptidyl peptidase 4 by targeting a substrate-specific secondary binding site

Dipeptidyl peptidase 4/CD26 (DP4) is a multifunctional serine protease liberating dipeptide from the N-terminus of (oligo)peptides which can modulate the activity of these peptides. The enzyme is involved in physiological processes such as blood glucose homeostasis and immune response. DP4 substrate specificity is characterized in detail using synthetic dipeptide derivatives. The specificity constant k(cat)/K(m) strongly depends on the amino acid in P?-position for proline, alanine, glycine and serine with 5.0 x 10? M?1 s?1, 1.8 x 10? M?1 s?1, 3.6 x 102 M?1 s?1, 1.1 x 102 M?1 s?1, respectively. By contrast, kinetic investigation of larger peptide substrates yields a different pattern. The specific activity of DP4 for neuropeptide Y (NPY) cleavage comprising a proline in P?-position is the same range as the k(cat)/K(m) values of NPY derivatives containing alanine or serine in P?-position with 4 x 10? M?1 s?1, 9.5 x 10? M?1 s?1 and 2.1 x 10? M?1 s?1, respectively. The proposed existence of an additional binding region outside the catalytic center is supported by measurements of peptide substrates with extended chain length. This 'secondary' binding site interaction depends on the amino acid sequence in P?'-P?'-position. Interactions with this binding site could be specifically blocked for substrates of the GRF/glucagon peptide family. By contrast, substrates not belonging to this peptide family and dipeptide derivative substrates that only bind to the catalytic center of DP4 were not inhibited. This more selective inhibition approach allows, for the first time, to distinguish between substrate families by substrate-discriminating inhibitors.