Role of Peptide Backbone Conformation on Biological Activity of Chemotactic Peptides

To investigate the role of peptide backbone conformation on the biological activity of chemotactic peptides, we synthesized a unique analog of N-formyl-Met-Leu-Phe-OH incorporating the C α,α disubstituted residue, dipropylglycine (Dpg) in place of Leu. The conformation of the stereochemically constrained Dpg analog was examined in the crystalline state by x-ray diffraction and in solution using NMR, IR, and CD methods. The secretagogue activity of the peptide on human neutrophils was determined and compared with that of a stereochemically constrained, folded type II β-turn analog incorporating 1-aminocyclohexanecarboxylic acid (Ac6c) at position 2 (f-Met- Ac6c -Phe-OMe), the parent peptide (f-Met-Leu-Phe-OH) and its methyl ester derivative (f-Met-Leu-Phe-OMe). In the solid state, the Dpg analog adopts an extended β-sheet-like structure with an intramolecular hydrogen bond between the NH and CO groups of the Dpg residue, thereby forming a fully extended (C5) conformation at position 2. The ϕ and ψ values for Met and Phe residues are significantly lower than the values expected for an ideal antiparallel beta conformation causing a twist in the extended backbone both at the N and C termini. Nuclear magnetic resonance studies suggest the presence of a significant population of the peptide molecules in an extended antiparallel β conformation and the involvement of Dpg NH in a C5 intramolecular hydrogen bond in solutions of deuterated chloroform and deuterated dimethyl sulfoxide. IR studies provide evidence for the presence of an intramolecular hydrogen bond in the molecule and the antiparallel extended conformation in chloroform solution. CD spectra in methanol, trifluoroethanol, and trimethyl phosphate indicate that the Dpg peptide shows slight conformational flexibility, whereas the folded Ac6c analog is quite rigid. The extended Dpg peptide consistently shows the highest activity in human peripheral blood neutrophils, being approximately 8 and 16 times more active than the parent peptide and the folded Ac6c analog, respectively. However, the finding that all four peptides have ED50 (the molar concentration of peptide to induce half-maximal enzyme release) values in the 10(-8)-10(-9) M range suggests that an induced fit mechanism may indeed be important in this ligand-receptor interaction. Moreover, it is also possible that alterations in the backbone conformation at the tripeptide level may not significantly alter the side chain topography and/or the accessibility of key functional groups important for interaction with the receptor

From one amino acid to another: tRNA-dependent amino acid biosynthesis

Aminoacyl-tRNAs (aa-tRNAs) are the essential substrates for translation. Most aa-tRNAs are formed by direct aminoacylation of tRNA catalyzed by aminoacyl-tRNA synthetases. However, a smaller number of aa-tRNAs (Asn-tRNA, Gln-tRNA, Cys-tRNA and Sec-tRNA) are made by synthesizing the amino acid on the tRNA by first attaching a non-cognate amino acid to the tRNA, which is then converted to the cognate one catalyzed by tRNA-dependent modifying enzymes. Asn-tRNA or Gln-tRNA formation in most prokaryotes requires amidation of Asp-tRNA or Glu-tRNA by amidotransferases that couple an amidase or an asparaginase to liberate ammonia with a tRNA-dependent kinase. Both archaeal and eukaryotic Sec-tRNA biosynthesis and Cys-tRNA synthesis in methanogens require O-phosophoseryl-tRNA formation. For tRNA-dependent Cys biosynthesis, O-phosphoseryl-tRNA synthetase directly attaches the amino acid to the tRNA which is then converted to Cys by Sep-tRNA: Cys-tRNA synthase. In Sec-tRNA synthesis, O-phosphoseryl-tRNA kinase phosphorylates Ser-tRNA to form the intermediate which is then modified to Sec-tRNA by Sep-tRNA:Sec-tRNA synthase. Complex formation between enzymes in the same pathway may protect the fidelity of protein synthesis. How these tRNA-dependent amino acid biosynthetic routes are integrated into overall metabolism may explain why they are still retained in so many organisms

Mitochondrial Ca²⁺ Uniporter haploinsufficiency enhances long-term potentiation at hippocampal mossy fibre synapses

Long-term changes in synaptic strength form the basis of learning and memory. These changes rely upon energy demanding mechanisms which are regulated by local Ca2+ signaling. Mitochondria are optimised for providing energy and buffering Ca2+. However, our understanding of the role of mitochondria in regulating synaptic plasticity is incomplete. Here we have used optical and electrophysiological techniques in cultured hippocampal neurons and ex vivo hippocampal slices from mice with haploinsufficiency of the mitochondrial Ca2+ uniporter (MCU+/-) to address whether reducing mitochondrial Ca2+ uptake alters synaptic transmission and plasticity. We found that cultured MCU+/- hippocampal neurons have impaired Ca2+ clearance, and consequently enhanced synaptic vesicle fusion at presynapses occupied by mitochondria. Furthermore, long-term potentiation (LTP) at mossy fibre (MF) synapses, a process which is dependent on presynaptic Ca2+ accumulation, is enhanced in MCU+/- slices. Our results reveal a previously unrecognized role for mitochondria in regulating presynaptic plasticity of a major excitatory pathway involved in learning and memory

Alpha-Synuclein Oligomers Interact with Metal Ions to Induce Oxidative Stress and Neuronal Death in Parkinson's Disease

Protein aggregation and oxidative stress are both key pathogenic processes in Parkinson's disease, although the mechanism by which misfolded proteins induce oxidative stress and neuronal death remains unknown. In this study, we describe how aggregation of alpha-synuclein (α-S) from its monomeric form to its soluble oligomeric state results in aberrant free radical production and neuronal toxicity

Multiscale, multimodal analysis of tumor heterogeneity in IDH1 mutant vs wild-type diffuse gliomas.

Glioma is recognized to be a highly heterogeneous CNS malignancy, whose diverse cellular composition and cellular interactions have not been well characterized. To gain new clinical- and biological-insights into the genetically-bifurcated IDH1 mutant (mt) vs wildtype (wt) forms of glioma, we integrated data from protein, genomic and MR imaging from 20 treatment-naïve glioma cases and 16 recurrent GBM cases. Multiplexed immunofluorescence (MxIF) was used to generate single cell data for 43 protein markers representing all cancer hallmarks, Genomic sequencing (exome and RNA (normal and tumor) and magnetic resonance imaging (MRI) quantitative features (protocols were T1-post, FLAIR and ADC) from whole tumor, peritumoral edema and enhancing core vs equivalent normal region were also collected from patients. Based on MxIF analysis, 85,767 cells (glioma cases) and 56,304 cells (GBM cases) were used to generate cell-level data for 24 biomarkers. K-means clustering was used to generate 7 distinct groups of cells with divergent biomarker profiles and deconvolution was used to assign RNA data into three classes. Spatial and molecular heterogeneity metrics were generated for the cell data. All features were compared between IDH mt and IDHwt patients and were finally combined to provide a holistic/integrated comparison. Protein expression by hallmark was generally lower in the IDHmt vs wt patients. Molecular and spatial heterogeneity scores for angiogenesis and cell invasion also differed between IDHmt and wt gliomas irrespective of prior treatment and tumor grade; these differences also persisted in the MR imaging features of peritumoral edema and contrast enhancement volumes. A coherent picture of enhanced angiogenesis in IDHwt tumors was derived from multiple platforms (genomic, proteomic and imaging) and scales from individual proteins to cell clusters and heterogeneity, as well as bulk tumor RNA and imaging features. Longer overall survival for IDH1mt glioma patients may reflect mutation-driven alterations in cellular, molecular, and spatial heterogeneity which manifest in discernable radiological manifestations

Substantiating a political public sphere in the Scottish press : a comparative analysis

This article uses content analysis to characterize the performance of the media in a national public sphere, by setting apart those qualities that typify internal press coverage of a political event. The article looks at the coverage of the 1999 devolved Scottish election from the day before the election until the day after. It uses a word count to measure the election material in Scottish newspapers the Herald, the Press and Journal and the Scotsman, and United Kingdom newspapers the Guardian, the Independent and The Times, and categorizes that material according to discourse type, day and page selection. The article finds a number of qualities that typify the Scottish sample in particular, and might be broadly indicative of a political public sphere in action. Firstly, and not unexpectedly, it finds that the Scottish newspapers carry significantly more election coverage. Just as tellingly, though, the article finds that the Scottish papers offer a greater proportion of advice and background information, in the form of opinion columns and feature articles. It also finds that the Scottish papers place a greater concentration of both informative and evaluative material in the period before the vote, consistent with their making a contribution to informed political action. Lastly, the article finds that the Scottish sample situates coverage nearer the front of the paper and places a greater proportion on recto pages. The article therefore argues that the Scottish papers display features that distinguish them from the UK papers, and are broadly consistent with their forming part of a deliberative public sphere, and suggests that these qualities might be explored as a means of judging future media performance

CSO Bolocam 1.1 mm continuum mapping of the Braid Nebula star formation region in Cygnus OB7

We present a 1.1 mm map of the Braid Nebula star formation region in Cygnus OB7 taken using Bolocam on the Caltech Submillimeter Observatory. Within the 1 deg2 covered by the map, we have detected 55 cold dust clumps all of which are new detections. A number of these clumps are coincident with IRAS point sources although the majority are not. Some of the previously studied optical/near-IR sources are detected at 1.1 mm. We estimate total dust/gas masses for the 55 clumps together with peak visual extinctions. We conclude that over the whole region, approximately 20% of the clumps are associated with IRAS sources suggesting that these are protostellar objects. The remaining 80% are classed as starless clumps. In addition, both FU Orionis (FUor) like objects in the field, the Braid Star and HH 381 IRS, are associated with strong millimeter emission. This implies that FUor eruptions can occur at very early stages of pre-main-sequence life. Finally, we determine that the cumulative clump mass function for the region is very similar to that found in both the Perseus and Ï? Ophiuchus star-forming regions. © 2011. The American Astronomical Society. All rights reserved

Mass Activated Droplet Sorting (MADS) Enables Highâ Throughput Screening of Enzymatic Reactions at Nanoliter Scale

Microfluidic droplet sorting enables the highâ throughput screening and selection of waterâ inâ oil microreactors at speeds and volumes unparalleled by traditional wellâ plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for highâ throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESIâ MS). Droplets are split, one portion is analyzed by ESIâ MS, and the second portion is sorted based on the MS result. Throughput of 0.7â samplesâ sâ 1 is achieved with 98â % accuracy using a selfâ correcting and adaptive sorting algorithm. We use the system to screen â 15â 000â samples in 6â h and demonstrate its utility by sorting 25â nL droplets containing transaminase expressed in vitro. Labelâ free ESIâ MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.A microfluidic system for sorting nanoliter droplets based on mass spectrometry is presented. Fully automated, labelâ free sorting at 0.7â samplesâ sâ 1 is achieved with 98â % accuracy. In vitro transcription and translation (ivTT) of a transaminase enzyme in ca.â 25â nL samples is demonstrated and samples are sorted on the basis of enzyme activity.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154315/1/anie201913203.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154315/2/anie201913203-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154315/3/anie201913203_am.pd