Funktionelle und strukturelle Charakterisierung des Ovastacins in vivo und in vitro

Die Metalloprotease Ovastacin, ein Vertreter der Astacin-Familie, wurde erstmals 2004 beschrieben. Im Ovar von Säugetieren ist Ovastacin-mRNA im Zeitfenster vom Stadium der Sekundärfollikel bis kurz nach der Befruchtung der Eizelle zu finden. Der Expressionsort und -zeitpunkt sowie die Sequenzähnlichkeit von über 60% mit sogenannten „Schlüpfenzymen“ (engl. hatching enzymes), die man in den Eizellen und Zygoten niederer Wirbeltiere und Wirbelloser gefunden hatte, ließen die Vermutung aufkommen, es könnte sich hier um das Säugerhomolog dieser Proteasen handeln. Generell lösen hatching Enzyme die derben embryonalen Hüllstrukturen (bei Säugern die Zona pellucida, ZP) beim Schlüpfvorgang auf. Die essentielle Bedeutung des Ovastacins für die Befruchtung wird durch die um ca. 30% reduzierte Fruchtbarkeit von Ovastacin defizienten Mäusen belegt. Hochinteressant war in diesem Zusammenhang die Entdeckung des Ovastacins in den Cortikalgranula der Oocyten sowie seine Fähigkeit, das Zona pellucida Protein 2 zu schneiden. Die dadurch bewirkte Verhärtung der Zona pellucida verhindert das Eindringen weiterer Spermien, das heißt sie baut eine Barriere gegen Polyspermie auf. Ziel dieser Arbeit war es, Belege für die physiologische Funktion des Ovastacins zu finden. Vor allem galt es, potentielle Aktivatoren zu identifizieren, da das Enzym wie alle Astacine als inaktive Vorstufe gebildet wird, die proteolytisch aktiviert werden muss. Zu diesem Zweck exprimierte ich rekombinantes Pro-Ovastacin in Insektenzellen. Aktivierungsstudien in vitro zeigten, dass ein saures Milieu zu einer Aktivierung führt, ohne die Abspaltung des Propeptids zu bewirken. Sequenzalignments und ein homologes Strukturmodell des Ovastacins wiesen auf Trypsin- oder Elastase-ähnliche Serinproteasen als potentielle Aktivierungsenzyme hin. Tatsächlich konnte mit diesen beiden Proteasetypen zum ersten Mal aktives Ovastacin aus Pro-Ovastacin erzeugt werden. Trypsin kommt als physiologischer Aktivator allerdings nicht in Betracht, da es bisher in keinem der Gewebe nachgewiesen werden konnte, in dem Ovastacin exprimiert wird. Die neutrophile Elastase dagegen konnte in der Leber, im Herz sowie im Blutplasma nachgewiesen werden. Mit Hilfe spezifischer Antikörper konnte das Herz als Expressionsort für Ovastacin bestätigt werden. Somit wäre Elastase ein potentieller physiologischer Aktivator von Ovastacin. Die Identifikation des Ovastacins in Geweben wie Leber, Herz, Nabelschnur und im Blutplasma weist auf eine Rolle der Protease in proteolytischen Netzwerken außerhalb der Spermien-Ei-Interaktion hin. Die Bedeutung der biologischen Kontrolle des Ovastacins bei der Befruchtung der Säugereizelle wird durch die Beobachtung untermauert, dass das Leberprotein Fetuin B als physiologischer Ovastacininhibitor fungiert und dadurch eine vorzeitige Verhärtung der Zona pellucida verhindert, die andernfalls die Penetration von Spermien prinzipiell verhindern würde.

Measurement of the e + e - pi + pi - cross section using initial state radiation at BESIII

The future goal of modern physics is the discovery of physics beyond the Standard Model. One of the most significant hints for New Physics can be seen in the anomalous magnetic moment of the muon - one of the most precise measured variables in modern physics and the main motivation of this work. This variable is associated with the coupling of the muon, an elementary particle, to an external electromagnetic field and is defined as a = (g - 2)/2, whereas g is the gyromagnetic factor of the muon. The muon anomaly has been measured with a relative accuracy of 0.5·10-6. However, a difference between the direct measurement and the Standard Model prediction of 3.6 standard deviations can be observed. This could be a hint for the existence of New Physics. Unfortunately, it is, yet, not significant enough to claim an observation and, thus, more precise measurements and calculations have to be performed.rnThe muon anomaly has three contributions, whereas the ones from quantum electrodynamics and weak interaction can be determined from perturbative calculations. This cannot be done in case of the hadronic contributions at low energies. The leading order contribution - the hadronic vacuum polarization - can be computed via a dispersion integral, which needs as input hadronic cross section measurements from electron-positron annihilations. Hence, it is essential for a precise prediction of the muon anomaly to measure these hadronic cross sections, σ(e+e-→hadrons), with high accuracy. With a contribution of more than 70%, the final state containing two charged pions is the most important one in this context.rnIn this thesis, a new measurement of the σ(e+e-→π+π-) cross section and the pion form factor is performed with an accuracy of 0.9% in the dominant ρ(770) resonance region between 600 and rn900 MeV at the BESIII experiment. The two-pion contribution to the leading-order (LO) hadronic vacuum polarization contribution to (g - 2) from the BESIII result, obtained in this work, is computed to be a(ππ,LO,600-900 MeV) = (368.2±2.5stat±3.3sys)·10-10. With the result presented in this thesis, we make an important contribution on the way to solve the (g - 2) puzzle.

Elevated level of metabotropic glutamate receptor 2/3 in the prefrontal cortex in major depression

Clinical, postmortem and preclinical research strongly implicates dysregulation of glutamatergic neurotransmission in major depressive disorder (MDD). Recently, metabotropic glutamate receptors (mGluRs) have been proposed as attractive targets for the discovery of novel therapeutic approaches against depression. The aim of this study was to examine mGluR2/3 protein levels in the prefrontal cortex (PFC) from depressed subjects. In addition, to test whether antidepressants influence mGluR2/3 expression we also studied levels of mGluR2/3 in fluoxetine-treated monkeys. Postmortem human prefrontal samples containing Brodmann's area 10 (BA10) were obtained from 11 depressed and 11 psychiatrically healthy controls. Male rhesus monkeys were treated chronically with fluoxetine (dose escalated to 3mg/kg, p.o.; n=7) or placebo (n=6) for 39 weeks. The mGluR2/3 immunoreactivity was investigated using Western blot method. There was a robust (+67%) increase in the expression of the mGlu2/3 protein in the PFC of depressed subjects relative to healthy controls. The expression of mGlu2/3 was unchanged in the PFC of monkeys treated with fluoxetine. Our findings provide the first evidence that mGluR2/3 is elevated in the PFC in MDD. This observation is consistent with reports showing that mGluR2/3 antagonists exhibit antidepressant-like activity in animal models and demonstrates that these receptors are promising targets for the discovery of novel antidepressants.

The Search for Low Energy Conformational Families of Small Peptides: Searching for Active Conformations of Small Peptides in the Absence of a Known Receptor

Breast cancer is the most common cancer among women. Tamoxifen is the preferred drug for estrogen receptor-positive breast cancer treatment, yet many of these cancers are intrinsically resistant to tamoxifen or acquire resistance during treatment. Therefore, scientists are searching for breast cancer drugs that have different molecular targets. Previous work revealed that 8-mer and cyclic 9-mer peptides inhibit breast cancer in mouse and rat model systems, interacting with an unknown receptor, while peptides smaller than eight amino acids did not inhibit breast cancer. We have shown that the use of replica exchange molecular dynamics predicts structure and dynamics of active peptides, leading to the discovery of smaller peptides with full biological activity. These simulations identified smaller peptide analogs with a conserved turn, a β-turn formed in the larger peptides. These analogs inhibit estrogen-dependent cell growth in a mouse uterine growth assay, a test showing reliable correlation with human breast cancer inhibition. We outline the computational methods that were tried and used with the experimental information that led to the successful completion of this research.

Computational approaches for the design of peptides with anti-breast cancer properties

Background: Breast cancer is the most common cancer among women. Tamoxifen is the preferred drug for estrogen receptor-positive breast cancer treatment, yet many of these cancers are intrinsically resistant to tamoxifen or acquire resistance during treatment. Therefore, scientists are searching for breast cancer drugs that have different molecular targets. Methodology: Recently, a computational approach was used to successfully design peptides that are new lead compounds against breast cancer. We used replica exchange molecular dynamics to predict the structure and dynamics of active peptides, leading to the discovery of smaller bioactive peptides. Conclusions: These analogs inhibit estrogen-dependent cell growth in a mouse uterine growth assay, a test showing reliable correlation with human breast cancer inhibition. We outline the computational methods that were tried and used along with the experimental information that led to the successful completion of this research.

The organization of memory for familiar songs

Investigated the organizing principles in memory for familiar songs in 2 experiments. It was hypothesized that individuals do not store and remember each song in isolation. Rather, there exists a rich system of relationships among tunes that can be revealed through similarity rating studies and memory tasks. One initial assumption was the division of relations among tunes into musical (e.g., tempo, rhythm) and nonmusical similarity. In Exp I, 20 undergraduates were asked to sort 60 familiar tunes into groups according to both musical and nonmusical criteria. Clustering analyses showed clear patterns of nonmusical similarity but few instances of musical similarity. Exp II, with 16 Ss, explored the psychological validity of the nonmusical relationships revealed in Exp I. A speeded verification task showed that songs similar to each other are confused more often than are distantly related songs. A free-recall task showed greater clustering for closely related songs than for distantly related ones. The relationship between these studies and studies of semantic memory is discussed. Also, the contribution of musical training and individual knowledge to the organization of the memory system is considered. (19 ref) (PsycINFO Database Record (c) 2012 APA, all rights reserved)

Shading Silhouettes of Smaller Ones

I am truly honored to have been given the amazing opportunity to create this original piece, this powerful journey through memory and emotive exploration of the loss of childhood. How do we feel about the loss of our child-self? Could we ever get them back? How long, how deep would one have to dig in the graveyards, the playgrounds of memory, to uncover what was buried there... to un-erase what waserased? shading silhouettes of smaller ones will ultimately encourage a reconnection with the Inner Child hidden inside all of us, as well as an intimate awareness of the adult version of the self by looking back to the smaller ones. The main inspiration for this piece is then of course, Inner Child Work. Most people may not be familiar with this therapeutic exploration of childhood... It wasimportant to me then, to present this concept in an imaginative, theatrical way, as a gift to you - a comprehensive and intensely moving gift. Speaking from experience, working on my Inner Child - my little Bianca - has been the most painful, frightening, yetrewarding and powerful experience within my personal life. Some people spend their entire lives trying to love themselves, to prove themselves, or be accepted. Some are too afraid to look back to where it all began. The characters within this piece will face thatfear... in a regression from the complexities of adulthood to the confusion of adolescence, all the way back to the wonder and bliss of childhood. They will reveal memories, of both joy and pain, love and abandonment, journeying backwards through time - through memory - through a playground - back to the beginning... We will enter a world where a push of a merry-go-round spins us to games of Truth or Dare after a high school dance at 16 - or the slam of a metal fence reminds us of the door Dad slammed in our face at 9 - where the sound of chain links swings us back to scrapping our knee by the sandbox at 5 This piece will attempt to connect everyone, both cast and audience, through a universal understanding and discussion of what it means to grow up, as well as a discovery of WHY we are the way we are - how experiences or relationships from our childhood have shaped our adult lives. We will attempt to challenge your honesty and nerve by inviting you to ask questions of yourselves, your past - to remember what it's like to have the innocence and hope of a child, to engage with and discover your Inner Child, to realize when or why you left them behind, and if you want to this magical part of yourself. It is my hope that you will join us in a collective journey - gather the courage to dig up the little kid you buried so long ago...* The creation, design, choreography, and direction for shading silhouettes of smaller ones mark the culminating experience of a year-long independent study in Theatre.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantitation of synthetic cannabinoids of the aminoalkylindole type and methanandamide in serum and its application to forensic samples

After the discovery of synthetic cannabimimetic substances in 'Spice'-like herbal mixtures marketed as 'incense' or 'plant fertilizer' the active compounds have been declared as controlled substances in several European countries. As expected, a monitoring of new herbal mixtures which continue to appear on the market revealed that shortly after control measures have been taken by legal authorities, other compounds were added to existing mixtures and to new products. Several compounds of the aminoalkylindole type have been detected so far in herbal mixtures but still their consumption cannot be detected by commonly used drug-screening procedures, encouraging drug users to substitute cannabis with those products. There is a increasing demand on the part of police authorities, hospitals and psychiatrists for detection and quantification of synthetic cannabinoids in biological samples originating from psychiatric inpatients, emergency units or assessment of fitness to drive. Therefore, a liquid chromatography-tandem mass spectrometry method after liquid-liquid extraction for the quantitation of JWH-015, JWH-018, JWH-073, JWH-081, JWH 200, JWH-250, WIN 55,212-2 and methanandamide and the detection of JWH-019 and JWH-020 in human serum has been developed and fully validated according to guidelines for forensic toxicological analyses. The method was successfully applied to 101 serum samples from 80 subjects provided by hospitals, detoxification and therapy centers, forensic psychiatric centers and police authorities. Fifty-seven samples or 56.4% were found positive for at least one aminoalkylindole. JWH-019, JWH-020, JWH-200, WIN 55,212-2 and methanandamide were not detected in any of the analyzed samples.

Chronicle of a death foretold: Plasmodium liver stage parasites decide on the fate of the host cell

Protozoan parasites of the genus Plasmodium are the causative agents of malaria. Despite more than 100 years of research, the complex life cycle of the parasite still bears many surprises and it is safe to say that understanding the biology of the pathogen will keep scientists busy for many years to come. Malaria research has mainly concentrated on the pathological blood stage of Plasmodium parasites, leaving us with many questions concerning parasite development within the mosquito and during the exo-erythrocytic stage in the vertebrate host. After the discovery of the Plasmodium liver stage in the middle of the last century, it remained understudied for many years but the realization that it represents a promising target for vaccination approaches has brought it back into focus. The last decade saw many new and exciting discoveries concerning the exo-erythrocytic stage and in this review we will discuss the highlights of the latest developments in the field.

Serodiagnosis of Echinococcus spp. infection: explorative selection of diagnostic antigens by peptide microarray

BACKGROUND: Production of native antigens for serodiagnosis of helminthic infections is laborious and hampered by batch-to-batch variation. For serodiagnosis of echinococcosis, especially cystic disease, most screening tests rely on crude or purified Echinococcus granulosus hydatid cyst fluid. To resolve limitations associated with native antigens in serological tests, the use of standardized and highly pure antigens produced by chemical synthesis offers considerable advantages, provided appropriate diagnostic sensitivity and specificity is achieved. METHODOLOGY/PRINCIPAL FINDINGS: Making use of the growing collection of genomic and proteomic data, we applied a set of bioinformatic selection criteria to a collection of protein sequences including conceptually translated nucleotide sequence data of two related tapeworms, Echinococcus multilocularis and Echinococcus granulosus. Our approach targeted alpha-helical coiled-coils and intrinsically unstructured regions of parasite proteins potentially exposed to the host immune system. From 6 proteins of E. multilocularis and 5 proteins of E. granulosus, 45 peptides between 24 and 30 amino acids in length were designed. These peptides were chemically synthesized, spotted on microarrays and screened for reactivity with sera from infected humans. Peptides reacting above the cut-off were validated in enzyme-linked immunosorbent assays (ELISA). Peptides identified failed to differentiate between E. multilocularis and E. granulosus infection. The peptide performing best reached 57% sensitivity and 94% specificity. This candidate derived from Echinococcus multilocularis antigen B8/1 and showed strong reactivity to sera from patients infected either with E. multilocularis or E. granulosus. CONCLUSIONS/SIGNIFICANCE: This study provides proof of principle for the discovery of diagnostically relevant peptides by bioinformatic selection complemented with screening on a high-throughput microarray platform. Our data showed that a single peptide cannot provide sufficient diagnostic sensitivity whereas pooling several peptide antigens improved sensitivity; thus combinations of several peptides may lead the way to new diagnostic tests that replace, or at least complement conventional immunodiagnosis of echinococcosis. Our strategy could prove useful for diagnostic developments in other pathogens.

Dynamic of ion channel expression at the plasma membrane of cardiomyocytes

Cardiac myocytes are characterized by distinct structural and functional entities involved in the generation and transmission of the action potential and the excitation-contraction coupling process. Key to their function is the specific organization of ion channels and transporters to and within distinct membrane domains, which supports the anisotropic propagation of the depolarization wave. This review addresses the current knowledge on the molecular actors regulating the distinct trafficking and targeting mechanisms of ion channels in the highly polarized cardiac myocyte. In addition to ubiquitous mechanisms shared by other excitable cells, cardiac myocytes show unique specialization, illustrated by the molecular organization of myocyte-myocyte contacts, e.g., the intercalated disc and the gap junction. Many factors contribute to the specialization of the cardiac sarcolemma and the functional expression of cardiac ion channels, including various anchoring proteins, motors, small GTPases, membrane lipids, and cholesterol. The discovery of genetic defects in some of these actors, leading to complex cardiac disorders, emphasizes the importance of trafficking and targeting of ion channels to cardiac function. A major challenge in the field is to understand how these and other actors work together in intact myocytes to fine-tune ion channel expression and control cardiac excitability.

Enzymatic Activity of Soybean Lipoxygenase-1 on Linoleoyl-Derivatized Agarose

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of linoleic acid to form 13(S) and 9(R) hydroperoxides. The manner in which substrates bind to the lipoxygenase family of enzymes is not known. It is believed fatty acid substrates may bind either with the aliphatic end first or with the carboxylate group facing the interior of the protein. This thesis tested a potential methyl-end first substrate binding mechanism by studying the activity of SBLO-1 to oxygenate immobilized linoleoyl residues attached to an insoluble polymer. Linoleic acid was attached to aminohexyl agarose in the presence of N-(3- dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and Nhydroxysuccinimide (NHS). The concentration of the covalently attached residues was facilitated by enriching linoleic acid with a small amount of the radioactive 14C-isotope. Functionalization yields of 3% available primary amines on the resin were obtained. Enzymatic oxygenation of the linoleoyl-residues was verified using the ferrous oxidation in xylenol orange (FOX) assay. Approximately 30% of the attached linoleoyl moieties were converted to hydroperoxides in the presence of SBLO-1. A disulfide-containing cleavable linker, cystamine, was used as part of an improved method to isolate the product in a facile manner. Cystamine was attached to NHS-activated agarose with approximately 5% overall functionalization yield of available functional groups. 14C-linoleic acid was successfully covalently linked to the cystamine moieties in the presence of EDC and NHS. The FOX assay verified the enzymatic oxygenation of the linoleoyl residues attached to cystamine-derivatized agarose. The isolation of the peroxide product was attempted in a series of extractions in organic solvents. The product was analyzed using GC/MS which did not show a new peak indicative of product. Further work is needed to successfully analyze the stereoand regiochemistry of the oxygenated product. The presence of the peroxides in this study indicated the linoleoyl residues behave as substrates of SBLO-1. It is unknown how bulky substrates bind to the active site; however, it is difficult to rationalize a carboxylate group-first binding mode. Discovery of the 13(S)-hydroperoxide product on the linoleoyl-agarose would support the claim of a potential methyl-end first binding mechanism.

Mechanical Properties of Plant Underground Storage Organs and Implications for Dietary Models of Early Hominins

The diet of early human ancestors has received renewed theoretical interest since the discovery of elevated d13C values in the enamel of Australopithecus africanus and Paranthropus robustus. As a result, the hominin diet is hypothesized to have included C4 grass or the tissues of animals which themselves consumed C4 grass. On mechanical grounds, such a diet is incompatible with the dental morphology and dental microwear of early hominins. Most inferences, particularly for Paranthropus, favor a diet of hard or mechanically resistant foods. This discrepancy has invigorated the longstanding hypothesis that hominins consumed plant underground storage organs (USOs). Plant USOs are attractive candidate foods because many bulbous grasses and cormous sedges use C4 photosynthesis. Yet mechanical data for USOs—or any putative hominin food—are scarcely known. To fill this empirical void we measured the mechanical properties of USOs from 98 plant species from across sub-Saharan Africa. We found that rhizomes were the most resistant to deformation and fracture, followed by tubers, corms, and bulbs. An important result of this study is that corms exhibited low toughness values (mean = 265.0 J m-2) and relatively high Young’s modulus values (mean = 4.9 MPa). This combination of properties fits many descriptions of the hominin diet as consisting of hard-brittle objects. When compared to corms, bulbs are tougher (mean = 325.0 J m-2) and less stiff (mean = 2.5 MPa). Again, this combination of traits resembles dietary inferences, especially for Australopithecus, which is predicted to have consumed soft-tough foods. Lastly, we observed the roasting behavior of Hadza hunter-gatherers and measured the effects of roasting on the toughness on undomesticated tubers. Our results support assumptions that roasting lessens the work of mastication, and, by inference, the cost of digestion. Together these findings provide the first mechanical basis for discussing the adaptive advantages of roasting tubers and the plausibility of USOs in the diet of early hominins.

Chemistry and Analysis of Phytocannabinoids and Other Cannabis Constituents

The Cannabis plant and its products consist of an enormous variety of chemicals. Some of the 483 compounds identified are unique to Cannabis, for example, the more than 60 cannabinoids, whereas the terpenes, with about 140 members forming the most abundant class, are widespread in the plant kingdom. The term “cannabinoids” [note: “ ” represents a group of C21 terpenophenolic compounds found until now uniquely in Cannabis sativa L. (1). As a consequence of the development of synthetic cannabinoids (e.g., nabilone [2], HU-211 [dexanabinol; ref. (3), or ajulemic acid [CT-3; ref. 4]) and the discovery of the chemically different endogenous cannabinoid receptor ligands (“endocannabinoids,” e.g., anandamide, 2-arachidonoylglycerol) (5,6), the term ’“phytocannabinoids’” was proposed for these particular Cannabis constituents (7).