Discovery of new scaffolds for GABA(A) receptor modulators from natural origin

Gamma-aminobutyric acid type A (GABAA) receptors are the major inhibitory neurotransmitter receptors in the central nervous system (CNS). These heteropentameric transmembrane proteins act as chloride ion channel upon activation by the endogenous ligand γ-amino butyric acid (GABA). Until now, 11 distinct GABAA receptor subtypes have been identified in the human brain. They differ in their subunit stoichiometry, tissue localization, functional characteristics, and pharmacological properties. Many CNS depressant drugs, such as the benzodiazepines exert their action via enhancement of the GABAergic neuronal inhibition. However, therapy may be accompanied by unwanted side-effects and specific clinical action is precluded due to the lack of GABAA receptor subtype selectivity. In a preliminary screen the lipophilic extracts of Piper nigrum fruits, Angelica pubescens roots, Acorus calamus roots, Biota orientalis leaves and twigs, and Kadsura longipedunculata fruits had shown positive GABAA receptor modulating activity in an in vitro functional, automated two-microelectrode voltage clamp assay with Xenopus laevis oocytes, which transiently expressed α1β2γ2S GABAA receptors. Aiming at the discovery of new scaffolds which act at the GABAA receptor, the active constituents of these five plant extracts were identified by means of an HPLC-based activity profiling approach. In total, we discovered 28 secondary metabolites with positive GABAA receptor modulating properties belonging to the structural classes of coumarins, monoterpenes, sesquiterpenes, diterpenes, phenylpropanes, piperamides, and lignans. Their structures were elucidated by a combination of powerful analytical methods such as HPLC-PDA-TOF-MS, highly sensitive microprobe NMR, and for chiral compounds, polarimetry and ECD. Determination of relative and absolute configuration was supported by conformational analysis and quantum chemical calculations. Furthermore, three yet unknown natural products could be identified. HPLC-based activity profiling with P. nigrum enabled the identification of 13 structurally related piperamides with minimum amount of extract. This allowed us to draw preliminary structure activity considerations for the scaffold of piperine, which was the main α1β2γ2S GABAA receptor modulator in this plant (EC50: 52.4 ± 9.4 μM, maximal stimulation of GABA induced chloride currents (IGABA) by 302% ± 27%). Sandaracopimaric acid and isopimaric acid from B. orientalis were tested for subtype selectivity at α1�3,5β1-3γ2S subtypes which revealed a comparatively high efficiency of both compounds at α2/3-subunit containing receptors. Additionally, sandaracopimaric acid exerted superior efficiency at receptors comprising β2-subunits. It showed EC50 values from 24.9 ± 6.3 μM to 82.2 ± 46.6 μM, and efficiencies ranging between 502% ± 56% to 1101% ± 98% potentiation of IGABA at the subtypes of investigation. A decrease of locomotor activity in the Open Field behavioral model was observed after intraperitoneal injection of 3 to 30 mg sandaracopimaric acid per kg bodyweight in mice. A trend towards anxiolytic-like activity could be observed with 1 and 3 mg/kg. Further “drug-like” GABAA receptor modulating scaffolds were discovered among the lignans from K. longipedunculata (potencies down to 12.8 ± 3.1 μM and efficiencies up to 886 ± 291% stimulation of IGABA) and among the sesquiterpenes from A. calamus (potencies down to 34.0 ± 6.7 μM and efficiencies up to 886 ± 105% stimulation of IGABA). These substances have potential for the further development as therapeutics acting at the GABAA receptor

MSUM Student Academic Conference

Minnesota State University Moorhead Student Academic Conference abstract book.https://red.mnstate.edu/sac-book/1013/thumbnail.jp

Medicinal Plants from Ancient Tradition as a Source for Matrix Proteases Inhibitors. Study of Correlation between Biological Activity and Phytochemical Profile

Considering the crucial involvement of matrix metalloproteinases’ (MMPs) misregulated activity in the pathogenesis of several degenerative diseases, this class of enzymes has been considered a highly active set of targets for the design of new therapeutic agents. However, the scant success of synthetic MMP inhibitors, largely due to the disappointing results obtained in both clinical and preclinical studies, makes medicinal plants a valuable source of new active compounds able to modulate MMPs activity. In this work, a consistent number of plants, selected on the base of an ethnobotanical research, were tested as inhibitors of collagenase, the founding member of the MMPs family. 1H-NMR-based metabolomic analysis combined with multivariate data treatment (PLS and OPLS) was used to correlate the biological activity to the phytochemical profiles, suggesting tannins as an important class of collagenase inhibitors. Thus, a tannin-removal procedure was developed, which allowed to prove this hypothesis and to identify another class of active metabolites, the glucuronide-conjugated flavonoids (especially quercetin-3-O-β-glucoronide), whose the plant Alchemilla vulgaris was found to be a good source. In another stage of the project, different varieties of tea were investigated as collagenase inhibitors, finding black tea samples particularly potent. Then, an OPLS model was developed with the aim of correlating the biological activity to the UV-Vis spectra of teas, showing that a high activity was related to absorption values in the range 350-440 nm. A subsequent fractionation of the most active tea sample was carried out, and this approach allowed to corroborate the results obtained by the metabolomic analysis. Considering that the absorbance measurement of an extract represents a cheap and simple procedure, the proposed method can be suitable, for instance, to select the best tea variety to be developed as an anti-wrinkles cosmetic or food supplement

Small business innovation research. Abstracts of completed 1987 phase 1 projects

Non-proprietary summaries of Phase 1 Small Business Innovation Research (SBIR) projects supported by NASA in the 1987 program year are given. Work in the areas of aeronautical propulsion, aerodynamics, acoustics, aircraft systems, materials and structures, teleoperators and robotics, computer sciences, information systems, spacecraft systems, spacecraft power supplies, spacecraft propulsion, bioastronautics, satellite communication, and space processing are covered

“Turning A New Leaf” Development of Spectroscopic Protocols to Study Plant Health

Improved methods to monitor plant health may be pivotal for future protection of crops from disease and environmental stressors. Epicuticular waxes, and macronutrients (K+ and Ca2+) hold important physiological functions within plants, and are therefore, excellent markers of plant health. This thesis has developed new spectroscopic analytical methods to directly image changes in epicuticular waxes and Ca2+ and K+ on plant leaves, for future application to monitor plant health

Research reports: 1990 NASA/ASEE Summer Faculty Fellowship Program

Reports on the research projects performed under the NASA/ASEE Summer Faculty Fellowship Program are presented. The program was conducted by The University of Alabama and MSFC during the period from June 4, 1990 through August 10, 1990. Some of the topics covered include: (1) Space Shuttles; (2) Space Station Freedom; (3) information systems; (4) materials and processes; (4) Space Shuttle main engine; (5) aerospace sciences; (6) mathematical models; (7) mission operations; (8) systems analysis and integration; (9) systems control; (10) structures and dynamics; (11) aerospace safety; and (12) remote sensin

Mass Spectrometric Investigation of the aging processes of ballpoint ink for the examination of questioned documents

In the forensic examination of documents the legitimacy of the age of an ink entry is often an essential question. Since its beginnings, the forensic questioned document field is concerned with the dating of inks. Inks aging processes follow complex paths and disagreements about the feasibility of actual methods raised worldwide among the scientific community. This controversy has been the starting point of the present work. Ball-point ink contains three main classes of compounds: solvents (50%), dyes (25%) and resins (25%). After the ink is deposited on paper, its composition begins to change qualitatively and quantitatively. While the dyes fade mainly through photochemistry processes, the aging behaviour of solvents are characterised by diffusion and evaporation processes. The aims were to actually study the aging processes of dyes and solvents found in ballpoint pens by modern mass spectrometric method in order to evaluate the potentiality and limitations of dynamic dating methods. Two major methods, Laser Desorption Ionisation - Mass Spectrometry (LDI-MS) for dyes and Gas Chromatography / Mass Spectrometry (GC/MS) for solvents were developed for this purpose and validated. The aging processes of reference substances were studied under defined storage conditions (light, heat, humidity). The same processes were investigated for the ballpoint dyes and solvents on the paper matrix. Therefore ballpoint ink strokes were exposed during several months to daylight and compared to strokes stored in the dark. On the one hand, small pieces of paper were cut, glued to a metallic sample holder with a carbon tape, and introduced into the ionisation chamber for direct analysis by LDI-MS. On the other hand, ink strokes were cut from the paper and extracted for subsequent GC/MS analysis. Moreover scanning microprobe LDI-MS was tested to microscopically visualize concentration profiles of ink dyes on the paper surface, i.e. it provides images of the chemical compound distribution. The determination of the writing sequence of crossing strokes was evaluated for questioned document expertises. Many factors influencing the fading of dye and the drying of solvents over time have been identified, to some extent evaluated. They can be divided in two mains categories: ballpoint ink composition and storage conditions of the document. Precise knowledge of these variables and their influence are necessary to deliver a scientific determination of the age of an ink entry. This work intend to give forensic scientists a better understanding of the complexity of the processes involved in the aging of a ballpoint ink matrix on a porous substrate such as paper. An overview of the ink dating issues is needed to comprehend how a dating method should be developed and validated in order to provide useful and accurate results. This has not been documented so far. Guidelines are proposed to compensate the limitations and set up valid ink dating methods. Any established dating method based on the fading of dyes or the drying of solvent has to be checked for reliability through indications of reproducibility, valid answers through blind testing and measurements errors lower than predictable variations.In den forensischen Wissenschaften ist die Bestimmung des Alters einer Kugelschreibereintragung (z.B. Kugelschreiberstrich) ein wichtiges Kriterium bei der Echtheitsprüfung von Dokumenten. Seit Beginn dieser Untersuchungen ist die Altersbestimmung eine wichtige und meist unbeantwortet Frage geblieben. Die forensischen Wissenschaftler streiten sich über die Möglichkeit und die Validierung von Datierungsmethoden. Diese Kontroverse ist der Angangspunkt dieser Arbeit gewesen. Kugelschreiberpasten bestehen aus drei Hauptkomponenten: Lösungsmitteln (50%), Farbstoffen (25%) und Harzen (25%). Nach dem Auftragen der Pasten auf Papier findet mit der Zeit eine qualitative und quantitative Veränderung der Zusammensetzung der Kugelschreibereintragung statt. Während Farbstoffe ausbleichen, d.h. vor allem photochemisch abgebaut werden, wird das Alterungsverhalten der Lösungsmittel durch den Verdampfungs- und Diffusionsprozess charakterisiert. In der vorgestellten Arbeit, sind die Alterungsprozesse von Farbstoffen und Lösungsmitteln mittels moderner massenspektrometrischen Methoden und deren möglichen Anwendung zur Altersbestimmung von Kugelschreibertinte untersucht worden. Zwei Hauptmethoden, Laser-Desorptions-/Ionisations-Massenspektrometrie (LDI-MS) für die Farbstoffen und Gas-Chromatographie-Massenspektrometrie (GC/MS) für die Lösungsmittel, sind für diesen Zweck methodisch weiterentwickelt und validiert worden. Die Alterungsprozesse von Referenzsubstanzen sind unter verschiedenen Bedingungen (Licht, Hitze, Feuchtigkeit) untersucht worden. Dieselben Prozesse sind dann für die Farbstoffen und Lösungsmittel auf der Papiermatrix verfolgt worden. Dazu wurden über mehrere Monate hinweg Eintragungen von mehreren Kugelschreibern dem normalen Sonnenlicht ausgesetzt und mit im Dunkeln gelagerten Proben verglichen. Dafür wurden einerseits kleine Stücke aus dem Papier ausgeschnitten und auf einem Probenteller befestigt, um dann LDI-MS Messungen direkt von den Tinten auf dem Papier durchzuführen. Andererseits wurden Kugelschreiberstriche für die GC-MS-Analyse aus dem Papier ausgeschnitten und anschließend extrahiert. Außerdem ist Ortaufgelöste LDI-MS getestet worden um die räumliche chemische Verteilung von Tinte auf dem Papier zu bestimmen. Die Reihenfolge zweier sich kreuzender Tintenlinien konnte auf diese Weise zur Echtheitsbestimmung des Dokumentes untersucht werden. Die Ergebnisse über Alterungsverhalten von Farbstoffen und Strichen von Kugelschreibern während einiger Jahre haben viele Faktoren offengelegt, die den Alterungsprozess beeinflussen. Zwei Hauptgruppen sind identifiziert worden: Die ursprüngliche Zusammensetzung der verwendeten Kugelschreiberpaste und die Lagerungsbedingungen des Dokumentes. Es ergibt eine umfangreiche, fundamentale und weitreichend nutzbare Studie der Alterung von Tinte, die bisher nicht existierte. Die Ergebnisse zeigen ausserdem auf welche Probleme ein Gutachter stoβen wird, wenn er eine Altersbestimmung durchführen will. Eine Strategie zur Entwicklung von Datierungsmethoden ist am Ende dieser Arbeit vorgeschlagen. Zuerst sollte eine ausführlische Tintendatenbank aufgebaut werden und zur Verfügung stehen. Gute und detaillierte Kenntiss der Alterungsprozesse aller vorkommenden Komponenten ist erforderlisch. Dazu dient die vorgestellte Arbeit als eine wissenschaftlische und protokollarische Vorgabe. Weitere Werte für bestimmte Alterungsparameter müssen trotzdem bestimmt werden, um eine mögliche Applikation zu entwicklen. Als letzter Schritt muss jede Methode zur Altersbestimmung unbedingt validiert werden

The 1985-86 NASA space/gravitational biology accomplishments

Individual Technical summaries of research projects of NASA's Space/Gravitational Biology Program are presented. This Program is concerned with using the unique characteristics of the space environment, particularly microgravity, as a tool to advance knowledge in the biological sciences; understanding how gravity has shaped and affected life on Earth; and understanding how the space environment affects both plant and animal species. The summaries for each project include a description of the research, a listing of the accomplishments, an explanation of the significance of the accomplishments, and a list of publications

Annual Report of the University, 1992-1993, Volumes 1-4

SIGNIFICANT DEVELOPMENTS Preparation, approval by President Peck, delivery to NMCHE of UNM\u27s response to House Memorials 38 and 25 (on minorities and women). Development and packaging of a presentation on minorities at UNM to Hispanic community people and organizations. Renewal of faculty instructional workload report and other information for use by President Peck and others in the President\u27s Council in testimony to the legislature on accountability by faculty. Significant workload and contributions to WICHE\u27s Diversity Project: - responses to long questionnaire - projected demographics - substitution for O. Forbes on planning for diversity Reprogramming of obsolete computer program of the University of Southern California\u27s Faculty Planning Model. Work remains incomplete. Support and staff work for University Planning Council, Faculty Senate Long Range Planning Committee, Senate President, Senate Budget Committee, Student Learning Outcomes Assessment Committee, Admissions and Registration Committee, Staff Council; Graduate Petition and grade Review Subcommittee Service to NMCHE\u27s Outcomes Assessment Advisory Group; NMCHE\u27s review group on diversity plans Service on Albuquerque Business/Education Compact Conducted several special data analyses to provide user outcome information for the Center for Academic Program Support (CAPS). Wrote reports to summarize analyses. Served in an advisory capacity to VP Zuniga Forbes for the two surveys (Campus Climate for Diversity, ACT Student Opinion Survey) and helped to draw the sample for the ACT survey. Conducted secondary analyses and prepared report of all analyses of the Freshman Survey (CIRP) for VP Zuniga Forbes. Gave presentation of CIRP findings to the Regents Subcommittee on Student Affairs. Conducted secondary analyses and prepared report of all analyses of the Campus Climate for Diversity Survey for VP Zuniga Forbes

Evaluation of X-ray Fluorescence Spectroscopy as a Tool for Element Analysis in Pea Seeds

X-ray fluorescence spectroscopy (XRF) is a powerful analytical tool for the determination of elemental composition of diverse materials. The principal aim of this study was to evaluate and characterize the utility and reliability of synchrotron-based XRF for use in element analysis of field pea seeds for quantification of macro- (K and Ca) and micro- (Mn, Fe, Cu, Zn, Se) elements. Pea seed samples were ground into fine flour and pellets were prepared to collect XRF peak intensities. Seventy-three pea seed samples were selected to cover the expected concentration ranges for each element for developing calibration curves by correlating the results from atomic absorption spectroscopy (AAS) reference method and XRF peak intensities. For all the calibration curves R2 values were above 0.8 except for K (0.5). XRF results were validated by a systematic comparison with data obtained from AAS on a set of 80 additional and independent pea seed samples (Validation Set). Element concentrations were also predicted using the fundamental parameter approach collectively for 153 samples. Limit of detection was calculated as low as 0.016 mg/kg for Se and 9.54 mg/kg for K. For Mn, Cu, Zn, Se, the XRF method was found to be not statistically different from AAS method at 95% confidence interval; furthermore, the bias between the methods was not significantly different from 0. Relative standard deviation (RSD) was less than 26% in XRF and range of recovery 75-165% for different elements. For the lower energy elements K and Ca, significant negative bias was observed (statistically different from 0) indicating underprediction by XRF method. The intercepts of the validation curve were -1460.3 and -61.27 for K and Ca respectively. Similar results were obtained with the fundamental parameter approach except for Fe for which significant bias of ~6 mg/kg was calculated. The intercept of validation curves was found to be not significantly different from 0 and B (the slope) was found to be not significantly different from 1. This leads to the conclusion that the results obtained using XRF and AAS were statistically not different from AAS method at 95% confidence interval. This study demonstrated that the XRF technique is a fast and reliable tool for element analysis, particularly for high energy elements and does not produce waste and requires no chemical reagents