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Ependymin Mechanism of Action: Full Length EPN VS Peptide CMX-8933
No full textEpendymin (EPN) is a goldfish neurotrophic factor (NTF) that is one of the most abundant secreted glycoprotein components of brain extracellular fluid (ECF) and cerebrospinal fluid. This protein was first discovered due to its enhanced turnover following learning events, but has since been found to function in other important cellular events such as long-term memory formation and optic nerve elongation (Shashoua, 1976; Shashoua, 1977; Shashoua, 1985). Goldfish EPN has several demonstrated effects on mammalian cells, and immuno-reactive EPN-like proteins have been observed in a variety of organisms ranging from invertebrates (Limulus) to mice.
Some NTFs have been shown to alleviate oxidative stress, one of the primary mediators of cell damage in neurodegenerative conditions. One mechanism by which they accomplish this is to increase cellular levels of anti-oxidative enzyme superoxide dismutase (SOD). In fact, our lab recently showed that a synthetic EPN fragment (CMX-8933) increases SOD mRNA and protein levels in rat primary cortical cultures (Parikh, 2003). Transgenic mice and rabbits that overexpress SOD are resistant to ischemia, while mice that lack SOD present with worse ischemic damage. Thus, due to this important SOD activating NTF-like feature of EPN may have potential therapeutic applications for treating neurodegenerative conditions such as Alzheimer's disease, Parkinson's or stroke.
Because full-length NTFs do not efficiently cross the blood brain barrier (BBB) when administered intravenously, our lab, in collaboration with Ceremedix, Inc. (Boston, MA), is interested in designing short peptides that mimic the action of full-length NTFs, especially EPN. Due to proteases that naturally exist in ECF, EPN is partially cleaved to release the 8 aa peptide KKETLQFR. Other brain proteins, like encephalins and endorphins, also release similar short active components, so we hypothesized that this 8 aa peptide may represent an active component of EPN. Indeed, our lab demonstrated that administration of this sequence in synthetic form (termed CMX-8933) to rat primary cortical cells increases cellular titers of SOD (Parkih, 2003).
This thesis was divided into three parts. The first part investigated which signal transduction pathway is responsible for CMX-8933's ability to upregulate SOD. Because our lab also showed that CMX-8933 activates the MAPK pathway (Hasson, 1998; El-Khishin, 1999; Adams et al., 2003) we hypothesized that CMX-8933 may use this pathway to upregulate SOD. Inhibition experiments were performed to test three known components of the MAPK pathway, and a member of an unrelated pathway. Six independent SOD immunoblot experiments demonstrated that pre-treatment of rat primary cortical cultures with specific inhibitors for the protein kinase-C family (PKC), protein tyrosine kinases (PTKs), or MEK protein kinases (MEKK), completely blocked (p = 0.0001) CMX-8933's average 15-fold upregulation of SOD. Thus, these three critical components of the MAPK pathway appear to be involved in the CMX-8933-induced upregulation of SOD. An inhibitor of transcription factor NF-êB in an unrelated pathway had no significant effect (p = 0.901).
The second part of this thesis tested whether treatment of rat primary cortical cultures with CMX-8933 increases the cellular titers of mRNAs related to translation. Previous observations indicated that treatment of these cells with with CMX-8933 induces neurite sprouting (Shashoua, unpublished), and that EPN plays a role in optic nerve elongation (Schmidt and Shashoua, 1988), two processes related to growth. So we hypothesized that EPN, or CMX-8933, may stimulate the transcriptioin of mRNAs related to growth. We tested mRNAs for translation factor EF-2, and ribosomal proteins S12 and L19 based on previous observations in our lab with hybridization arrays (Parikh, 2003). RT-PCR experiments indicated that treatment of rat primary cortical cultures with 10 ng/ml CMX-8933 for 5 hrs increased the mRNAs for S12 an average of 12-fold relative to untreated cultures (N = 3, p = 0.02), L19 an average of 9-fold (N = 3, p = 0.048), and EF-2 an average of 11-fold (N = 3, p = 0.045). Levels of housekeeper polyubiquitin remained unchanged. Thus 3 gene products related to growth are indeed upregulated by CMX-8933.
The third part of this thesis investigated the SOD stimulatory effects of full-length EPN versus its cleavage product CMX-8933. Previous studies showed that full-length NTFs BDNF and NGF upregulate SOD in neuronal cells. Because CMX-8933 upregulates SOD, maybe full-length EPN does too. We hypothesized that if CMX-8933 represents the receptor-binding domain of full-length EPN, that full-length EPN may show the same stimulatory effects as CMX-8933, and may upregulate SOD. Extracellular fluid (ECF) was prepared from goldfish brains, the traditional source for isolating EPN. Analysis of the ECF on protein gels demonstrated the presence of a complex protein pattern dominated by two bands at 37,000 and 31,000 daltons, the known sizes of EPN-â (glycosylated) and EPN-ã (non-glycosylated), respectively. Immunoblots performed with EPN antibody ""Sheila"" (directed against the C-terminal end of EPN, Shashoua and Moore, 1978) confirmed the identity of these two ECF bands as EPN. Cultured mouse Nb2a neuronal cells were treated in six independent experiments with 12 ìg/ml ECF protein for 5 hrs, and whole cell lysates were tested for levels of SOD by immunoblots. This ECF treatment of neuronal cells produced a mean 4-fold increase in SOD levels (p = 0.007), supporting our hypothesis.
However, since ECF is a complex mixture, this data did not show which ECF component was resonsible for the SOD signal. Since ECF is known to contain CMX-8933 EPN cleavage product, which by itself can upregulate SOD, it is possible CMX-8933 was responsible for the signal, not full-length EPN. To address this issue, microdialysis was performed using an 8,000 dalton MWCO membrane to remove low MW components from the ECF (including CMX-8933, MW = 1149), leaving EPN-â (MW 37,000) and EPN-ã (MW 31,000) present in the dialyzed ECF. Four independent experiments indicated no significant difference (p = 0.116) between dialyzed versus non-dialyzed ECF for activating SOD. Thus, CMX-8933 does not appear to be responsible for ECF's ability to increase SOD, but instead, high MW molecules (including full-length EPN) appear to be the active components. Altogether, the data from this thesis extends our knowledge of the mechanism of action of both full-length EPN and its cleavage product CMX-8933
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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AP-1 Is Required For CMX-8933-Induced SOD Upregulation And Is Translocated In Response To A Human EPN Mimetic
No full textEpendymin (EPN) is a neurotrophic factor (NTF) that functions in goldfish long-term memory formation and optic nerve elongation (Shashoua, 1976; Shashoua, 1977; Shashoua, 1985). Goldfish EPN, or CMX-8933 (a short goldfish EPN mimetic studied by our lab), surprisingly have several demonstrated effects on mammalian cells, including neuroregenerative effects in a rat stroke model (Shashoua et al, 2003), and the activation of therapeutic superoxide dismutase (SOD) (Parikh, 2003) and transcription factor AP-1 (Adams et al, 2003) in mouse neuroblastoma cells or rat primary cortical neuronal cultures. Among its various functions, AP-1 can function as a master switch in long-term memory consolidation (Sanyal et al, 2002), so it may be a key event in EPN’s mechanism of action. AP-1 activation is also a characteristic associated with other full-sized neurotrophic factors, including nerve growth factor and brain-derived nerve growth factor.
This thesis was divided into three parts. The purpose of part I was to determine whether our previously observed upregulation of SOD by CMX-8933 is dependent upon (or merely concurrent with) AP-1 activation. Four independent SOD immunoblot experiments demonstrated that pre-treatment of rat primary cortical cultures with trifluoromethyl pyrimidine carboxylate (TFPC), a specific inhibitor of AP-1, significantly (p = 0.0004) decreased cellular levels of SOD by 67% at its IC50 concentration of 1 ìM, and completely inhibited the upregulation at 10 and 100 ìM concentrations. Thus, the CMX-8933-induced upregulation of SOD appears to depend (directly or indirectly) on AP-1 activation.
Part II of this thesis included the use of bioinformatics to re-verify exciting recent observations that EPN-like proteins exist in mammals, termed mammalian-ependymin-related proteins or MERPs (Apostolopoulos et al, 2001). If our analyses were convincing, human EPN mimetics would then be designed and tested for AP-1 activation. Computer alignments and hydropathy plots performed with EPN amino acid sequences deduced from gene entries in GenBank verified the existence of mammalian homologs containing highly conserved domains with fish EPN’s, suggesting the possibilities of similar protein conformation and function. Two human EPN mimetics were designed, hEPN-1 (8 aa long, corresponding to the same region as CMX-8933) and hEPN-2 (14 aa long, containing CMX-8933 and 6 upstream aa). Several mimetic doses were tested on mouse Neuro-2a cultures for nuclear translocation of c-Jun and c-Fos proteins (comprising the AP-1 particle upregulated by fish CMX-8933). Seven independent c-Jun immunoblot experiments, and five c-Fos experiments, demonstrated a strong (as high as 25-fold) dose-dependent increase in the nuclear titers of the AP-1 proteins. Both peptides had statistically equivalent effects. Thus, human EPN appears to exist, and two mimetics derived from its sequence appear to be biologically active against mouse neuroblastoma cells. Since hEPN-1 and -2 have only a few residues in common with CMX-8933, we hypothesize that the mimetic shape rather than sequence may be important for biological activity.
In part III of this thesis, the biological effects of hEPN-1 and hEPN-2 on mouse Neuro-2a cells were studied further using RT-PCR to analyze potential increases in specific mRNAs. mRNAs related to growth, energy production, and protein translation were tested since previous data in our lab (Kaska, 2003) indicated mRNAs for translational elongation factor-2 (EF-2), and ribosomal proteins L19 and S12 were upregulated in rat primary cortical cultures by fish mimetic CMX-8933 (Kaska, 2003). Treatment of Neuro-2a cells with 1.0 ìg/ml hEPN-1 (the highest dose tested for the AP-1 translocation experiments) for 24 hrs appeared to increase (N = 1) mRNAs for ATP Synthase-C, ribosomal protein L19, and translational EF-2, relative to the levels of housekeeper polyubiquitin. Thus hEPN-1 may be involved in processes related to growth.
Altogether, the data from this thesis extends our knowledge of fish EPN mimetic CMX-8933 (showing that its induction of SOD requires AP-1), demonstrates that human EPN may exist (bioinformatics), shows that two human EPN mimetics are biologically active (induce AP-1 translocation), and shows that one mimetic hEPN-1 may activate several mRNAs related to growth in mouse Neuro-2a cells
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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