27 research outputs found
Phage display-derived inhibitor of the essential cell wall biosynthesis enzyme MurF
No full textBackground
To develop antibacterial agents having novel modes of action against bacterial cell wall biosynthesis, we targeted the essential MurF enzyme of the antibiotic resistant pathogen Pseudomonas aeruginosa. MurF catalyzes the formation of a peptide bond between D-Alanyl-D-Alanine (D-Ala-D-Ala) and the cell wall precursor uridine 5'-diphosphoryl N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-diaminopimelic acid (UDP-MurNAc-Ala-Glu-meso-A2pm) with the concomitant hydrolysis of ATP to ADP and inorganic phosphate, yielding UDP-N-acetylmuramyl-pentapeptide. As MurF acts on a dipeptide, we exploited a phage display approach to identify peptide ligands having high binding affinities for the enzyme.
Results
Screening of a phage display 12-mer library using purified P. aeruginosa MurF yielded to the identification of the MurFp1 peptide. The MurF substrate UDP-MurNAc-Ala-Glumeso-A2pm was synthesized and used to develop a sensitive spectrophotometric assay to quantify MurF kinetics and inhibition. MurFp1 acted as a weak, time-dependent inhibitor of MurF activity but was a potent inhibitor when MurF was pre-incubated with UDP-MurNAc-Ala-Glu-meso-A2pm or ATP. In contrast, adding the substrate D-Ala-D-Ala during the pre-incubation nullified the inhibition. The IC50 value of MurFp1 was evaluated at 250 μM, and the Ki was established at 420 μM with respect to the mixed type of inhibition against D-Ala-D-Ala.
Conclusion
MurFp1 exerts its inhibitory action by interfering with the utilization of D-Ala-D-Ala by the MurF amide ligase enzyme. We propose that MurFp1 exploits UDP-MurNAc-Ala-Glu-meso-A2pm-induced structural changes for better interaction with the enzyme. We present the first peptide inhibitor of MurF, an enzyme that should be exploited as a target for antimicrobial drug development
Effects of temporary inactivation of dorsal hippocampus on explicitly nonspatial, unimodal, contextual fear learning
No full textSeveral studies have reported that dorsal hippocampal damage attenuates the acquisition (Kim et al., 1993; Phillips & LeDoux, 1992; Young et al., 1994) or expression (Anagnostaras et al., 1999; Holt & Maren, 1999) of recently acquired contextual fear conditioning. "Context" is often operationalized as the conditioning chamber in which CS-US pairings occurred. However, the hippocampus is known to participate in spatial learning, presenting interpretative difficulties regarding the role of dorsal hippocampus in learning and memory. The current study examined the effects of temporary inactivation of DH on freezing, rearing, ambulating, grooming, and whisking behavior in an explicitly nonspatial contextual fear conditioning paradigm, where olfactory stimuli served as temporally and spatially diffuse contexts. Results indicate that temporary inactivation of DH produced both anterograde and retrograde deficits in contextually conditioned freezing, while sparing the acquisition and expression of freezing to a discrete auditory CS. Further, animals with DH inactivation froze modestly and similarly to the unsafe and safe contextual stimuli, while intact animals froze robustly to the unsafe, but not the safe, contextual stimulus. These data indicate that there is a decidedly nonspatial component to the role of DH in contextual conditioning, and suggest that olfactory contextual conditioning is a fruitful means of further exploring this function.M.S.Includes bibliographical references (p. 32-36)
DNA fusion gene vaccination mobilizes effective anti-leukemic cytotoxic T lymphocytes from a tolerized repertoire
No full textThe majority of known human tumor-associated antigens derive from non-mutated self proteins. T cell tolerance, essential to prevent autoimmunity, must therefore be cautiously circumvented to generate cytotoxic T cell responses against these targets. Our strategy uses DNA fusion vaccines to activate high levels of peptide-specific CTL. Key foreign sequences from tetanus toxin activate tolerance-breaking CD4+ T cell help. Candidate MHC class Ibinding tumor peptide sequences are fused to the C terminus for optimal processing and presentation. To model performance against a leukemia-associated antigen in a tolerized setting, we constructed a fusion vaccine encoding an immunodominant CTL epitopederived from Friend murine leukemia virus gag protein (FMuLVgag) and vaccinated tolerant FMuLVgag-transgenic (gag-Tg) mice. Vaccination with the construct induced epitopespecificIFN-c-producing CD8+ T cells in normal and gag-Tg mice. The frequency and avidity of activated cells were reduced in gag-Tg mice, and no autoimmune injury resulted. However, these CD8+ T cells did exhibit gag-specific cytotoxicity in vitro and in vivo. Also, epitope-specific CTL killed FBL-3 leukemia cells expressing endogenous FMuLVgag antigen and protected against leukemia challenge in vivo. These results demonstrate a simple strategy to engage anti-microbial T cell help to activate epitope-specific polyclonal CD8+ T cell responses from a residual tolerized repertoire
Pictures, power and the polity: a vision of the political images of the early Dutch Republic. [In two volumes]
No full textThe Dutch Revolt (c. 1568-1648) led to the establishment of a new state in the northern provinces of the old Habsburg Netherlands. This new polity confronted intense hostility from Habsburg dynastic interests. It sustained itself militarily against these interests, and extended its power globally. In addition it developed a remarkable and wealthy mercantile culture. However configurations of power in the new state differed radically from those within the surrounding monarchies, and its political texts remain problematic.Thus there is no dynamic political theory to match the reality of its might. However, one of the remarkable features of its culture was the unprecedented output of pictorial art, including thousands of political prints. Therefore, this thesis addresses the issue of power in the Republic on the basis of pictorial evidence, using a combination of three routes. First, instead of examining evidence made up of texts, it was decided to use a range of political imagery, largely political prints, to serve as primary sources, inverting the usual practice of alluding to images from an argument based on texts.Second, there is a requirement upon historians for a systematic approach to primary sources, allowing argument to be tightly referenced. However, imagery is not subject to the usual methods (footnoting chapters and pages for example), so a methodology was developed which incorporates digitally modelled representations of the prints.This was based upon work undertaken by Gerhard Jaritz at the Instituts für Realienkunde des Mittelalters und der Frühen Neuzeit in Austria. Thirdly, prompted by the doubts of several scholars about the utility of conventional political theories in the context of the Dutch Republic, the work of Michel Foucault, in particular his prescription for the study of power, has been adapted and used as ananalytical framework in which to discuss the sources.The thesis demonstrates the systematic exploitation of pictorial sources in the context of historical study. It demonstrates the advantages and limitations of digital models and computer analysis. On the basis of these novel methodologies, the thesis summarises a thorough exploration of a range of political imagery. It also highlights the extraordinary success of a particular image of the Revolt, the Tyranny of Alva. On the basis of the evidence examined, it also demonstrates that there was a profound antipathy towards monarchic, 'top down' power in the early Republic, and argues that power there was more easily diagrammed than textualised
Time course of dorsal and ventral hippocampal involvement in the expression of trace fear conditioning
No full textIt is becoming increasingly clear that the time-dependent involvement of the hippocampus in the recall of acquired behaviors is more complicated than once thought. Several early studies demonstrated that hippocampal damage attenuates the expression of recent, but not remotely trained tasks. By contrast, an emerging body of evidence has shown that damage to, or inactivation of, the hippocampus impairs recall across a wide range of training-testing intervals. Collectively, these data suggest that the time course of hippocampal involvement in the storage or recall of previously-acquired memories differs according to hippocampal subregion and the particular learning task under consideration. The present study examined the contributions of dorsal (DH) and ventral (VH) hippocampus to the expression of previously-acquired trace fear conditioning, a form of Pavlovian conditioning in which the presentation of an initially neutral cue or cues and a subsequent aversive stimulus is separated by a no-stimulus (trace) interval. Specifically, either the GABA-A agonist muscimol or saline was infused into the DH or VH prior to testing 1, 7, 28, or 42 days after trace fear conditioning. The results revealed a marked dissociation: pre-testing inactivation of DH failed to impair performance at any time-point, while pre-testing inactivation of VH impaired performance at all time-points. Importantly, pre-testing inactivation of VH had no effect on the performance during testing of previously-acquired delay conditioning. Collectively, these data suggest that VH, but not DH, remains a neuroanatomical locus critical to the recall or expression of trace fear conditioning over an extended period of time.M.S.Includes bibliographical referencesIncludes vitaby David Henry Co
The emerging role for bacteria in lignin degradation and bio-product formation
No full textThe microbial degradation of lignin has been well studied in white-rot and brown-rot fungi, but is much less well studied in bacteria. Recent published work suggests that a range of soil bacteria, often aromatic-degrading bacteria, are able to break down lignin. The enzymology of bacterial lignin breakdown is currently not well understood, but extracellular peroxidase and laccase enzymes appear to be involved. There are also reports of aromatic-degrading bacteria isolated from termite guts, though there are conflicting reports on the ability of termite gut micro-organisms to break down lignin. If biocatalytic routes for lignin breakdown could be developed, then lignin represents a potentially rich source of renewable aromatic chemicals
Simplified novel muraymycin analogues ; using a serine template strategy for linking key pharmacophores
No full textThe present status of antibiotic research requires the urgent invention of novel agents that act on multidrug-resistant bacteria. The World Health Organization has classified antibiotic-resistant bacteria into critical, high and medium priority according to the urgency of need for new antibiotics. Naturally occurring uridine-derived "nucleoside antibiotics" have shown promising activity against numerous priority resistant organisms by inhibiting the transmembrane protein MraY (translocase I), which is yet to be explored in a clinical context. The catalytic activity of MraY is an essential process for bacterial cell viability and growth including that of priority organisms. Muraymycins are one subclass of naturally occurring MraY inhibitors. Despite having potent antibiotic properties, the structural complexity of muraymycins advocates for simplified analogues as potential lead structures. Herein, we report a systematic structure-activity relationship (SAR) study of serine template-linked, simplified muraymycin-type analogues. This preliminary SAR lead study of serine template analogues successfully revealed that the complex structure of naturally occurring muraymycins could be easily simplified to afford bioactive scaffolds against resistant priority organisms. This study will pave the way for the development of novel antibacterial lead compounds based on a simplified serine template. [Abstract copyright: © 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Pharmaceutical applications of lignin-derived chemicals and lignin-based materials : linking lignin source and processing with clinical indication
No full textLignocellulosic biomass is one of the most abundant bioresources on Earth. Over recent decades, various valorisation techniques have been developed to produce value-added products from the cellulosic and hemicellulosic fractions of this biomass. Lignin is the third major component accounting for 10–30% (w/w). However, it currently remains a largely unused fraction due to its recalcitrance and complex structure. The increase in the global demand for lignocellulosic biomass, for energy and chemical production, is increasing the amount of waste lignin available. Approaches to date for valorizing this renewable but heterogeneous chemical resource have mainly focused on production of materials and fine chemicals. Greater value could be gained by developing higher value pharmaceutical applications which would help to improve integrated biorefinery economics. In this review, different lignin extraction methods, such as organosolv and ionic liquid, and the properties and potential of the extracted chemical building blocks are first summarized with respect to pharmaceutical use. The review then discusses the many recent advances made regarding the medical or therapeutic potential of lignin-derived materials such as antimicrobial, antiviral, and antitumor compounds and in controlled drug delivery. The aim is to draw out the link between the source and the processing of the biomass and potential clinical applications. We then highlight four key areas for future research if therapeutic applications of lignin-derived products are to become commercially viable. These relate to the availability and processing of lignocellulosic biomass, technologies for the purification of specific compounds, enhancements in process yield, and progression to human clinical trials
Organically bound iodine as a bottom-water redox proxy : preliminary validation and application
No full text© The Author(s), 2017. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Chemical Geology 457 (2017): 95-106, doi:10.1016/j.chemgeo.2017.03.016.Carbonate-associated iodine (I/Ca) has been used as a proxy of local, upper-ocean redox conditions, and has successfully demonstrated highly dynamic spatial and temporal patterns across different time scales of Earth history. To further explore the utility of iodine as a paleo-environmental proxy, we present here a new method of extracting organically bound iodine (Iorg) from shale using volumes of samples on the order of tens of milligrams, thus offering the potential for high-resolution work across thin shale beds. The ratio of Iorg to total organic carbon (I/TOC) in modern surface and subsurface sediments decreases with decreasing bottom-water oxygen, which may be used to reconstruct paleo-redox changes.
As a proof of concept, we evaluate the I/TOC proxy in Holocene sediments from the Baltic Sea, Landsort Deep (IODP 347) and discuss those data within a framework of additional independent redox proxies, e.g., iron speciation and [Mo]. The results imply that I/TOC may be sensitive to hypoxic–suboxic conditions, complementary to proxies sensitive to more reducing, anoxic–euxinic conditions. Then, we test the usage of I/TOC in sediments deposited during Late Cretaceous, Cenomanian–Turonian Oceanic Anoxic Event (OAE) 2 from ~ 94 million years ago (Ma). We generated I/TOC and Iorg records from six OAE 2 sections: Tarfaya (Morocco), Furlo (central Italy), Demerara Rise (western equatorial Atlantic), Cape Verde Basin (eastern equatorial Atlantic), South Ferriby (UK), and Kerguelen Plateau (southern Indian Ocean), which provide a broad spatial coverage. Generally, I/TOC decreases over the interval recorded by the positive carbon-isotope excursion, the global signature of OAE 2, suggesting an expansion of more reducing bottom-water conditions and consistent with independent constraints from iron speciation and redox-sensitive trace-metals (e.g., Mo). Relatively higher I/TOC values (thus more oxic conditions) are recorded at two high latitude sites for OAE 2, supporting previous model simulations (cGENIE) that indicated higher bottom water oxygen concentrations in these regions. Our results also indicate that organic-rich and oxygenated seafloors are likely a major sink of iodine and correspondingly influence its global seawater inventory.XZ, WL and ZL are supported by NSF EAR 1349252. DH and TWL acknowledge support from
the Geobiology and Low-temperature Geochemistry (GG) Program of NSF. DH would like to
acknowledge a Schlanger Ocean Drilling Fellowship
