The timely administration of antivenom is the most effective method currently available to reduce the burden of snakebite envenoming (SBE), a neglected tropical disease that most often affects rural agricultural global populations. There is increasing interest in the development of adjunctive small molecule and biologic therapeutics that target the most problematic venom components to bridge the time-gap between initial SBE and the administration antivenom. Unique combinations of these therapeutics could provide relief from the toxic effects of regional groupings of medically relevant snake species. The application a PRISMA/PICO literature search methodology demonstrated an increasing interest in the rapid administration of therapies to improve patient symptoms and outcomes after SBE. Advice from expert interviews and considerations regarding the potential routes of therapy administration, anatomical bite location, and species-specific venom delivery have provided a framework to identify ideal metrics and potential hurdles for the development of a field-based medical device that could be used immediately after SBE to deliver adjunctive therapies. The use of subcutaneous (SC) or intramuscular (IM) injection were identified as potential routes of administration of both small molecule and biologic therapies. The development of a field-based medical device for the delivery of adjunctive SBE therapies presents unique challenges that will require a collaborative and transdisciplinary approach to be successful.

A variety of opossum species are resistant to snake venoms due to the presence of antihemorrhagic and antimyotoxic acidic serum glycoproteins that inhibit several toxic venom components. Two virtually identical antihemorrhagic proteins isolated from either the North American opossum (D. virginiana) or the South American big-eared opossum (D. aurita), termed oprin or DM43 respectively, inhibit specific snake venom metalloproteinases (SVMPs). A better understanding of the structure of these proteins may provide useful insight to determine their mechanism of action and for the development of therapeutics against the global health concern of snake-bite envenomation. The aim of this work is to produce a recombinant snake venom metalloproteinase inhibitor (SVMPI) similar to the above opossum proteins in Escherichia coli and determine if this bacterially produced protein inhibits the proteolytic properties of Western Diamondback rattlesnake (C. atrox) venom. The resulting heterologous SVMPI was produced with either a 6-Histidine or maltose binding protein (MBP) affinity tag on either the C-terminus or N-terminus of the protein, respectively. The presence of the solubility enhancing MBP affinity tag resulted in significantly more soluble protein expression. The inhibitory activity was measured using two complementary assays and the MBP labeled SVMPI showed 7-fold less activity as compared to the 6-Histidine labeled SVMPI. Thus, the bacterially derived SVMPI with an unlabeled N-terminus showed high inhibitory activity (IC50 = 4.5 μM). The use of a solubility enhancing MBP fusion protein construct appears to be a productive way to express sufficient quantities of this mammalian protein in E. coli for further study.

Understanding how to perform an enzyme assay is a critical learning skill in the undergraduate biochemistry curriculum. Students in biochemistry typically have been exposed to the use of NMR spectroscopy as a tool to determine chemical structure, but rarely are they exposed to the utility of NMR to evaluate enzyme kinetics. Furthermore, coverage of NMR experiments utilizing "alternative nuclei", such as 15 N, 19 F, and 31 P may be neglected. Herein we report a simple 31 P NMR tube experiment that allows students to examine the enzyme kinetics and equilibrium constant of the reaction catalyzed by pyruvate kinase. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(6):509-514, 2017.

To determine whether natural recovery was occurring in a depositional area of the St. Marys River (Ontario, Canada) known as East Bellevue Marine Park (EBMP), sediment was collected from two depth ranges, 0–5 cm and 0–10 cm, and subjected to a series of laboratory toxicity tests and chemical analysis. Toxicological responses (survival, growth, reproduction, development) of four benthic invertebrates and the fathead minnow were compared at test vs. reference sites using univariate and multivariate (ordination) techniques. Temporal trends in sediment chemistry and invertebrate toxicity were examined with time series data from 2008 through to 2018. Polycyclic aromatic hydrocarbons (PAHs; ≤ 37 mg/kg) and petroleum hydrocarbons (PHCs; ≤ 6266 mg/kg) were elevated in EBMP compared to reference sites (PAHs, ≤ 1.6 mg/kg; PHCs ≤ 180 mg/kg). Comparatively, the 0–5 cm sediment layer had lower concentrations of all contaminants than the 0–10 cm layer at three of four test sites. Over time, contaminant concentrations have mostly remained stable or have decreased. There were no significant differences in survival, growth, or development of the larval fish in EBMP compared to the upstream reference sites, and no differences between sampling depths. However, most EBMP sediments were toxic to invertebrates, driven by reduced reproduction by the worm Tubifex and reduced survival by the amphipod Hyalella. Among habitat variables, a combination of different classes of compounds based on ordination scores (PHCs, oil and grease, metals) was most strongly correlated to toxicological response. There was little to no difference in toxicity between sampling depths based on integrated endpoint response; however, individual endpoints showed mostly greater toxicity from exposure to the 0–10 cm layer. Over time, toxicity has mostly remained stable or showed improvement. These results provided some positive indications that gradual natural recovery is occurring in EBMP.

The St. Marys River is the sole surface outlet from Lake Superior, flowing over 100 km to Lake Huron, and forming part of the international border between Canada (Ontario) and the United States (MI). The river system has been severely impacted by modifications to the hydrology as well as local industrial and municipal discharges, and was designated in 1985 as an Area of Concern (AOC). Since that time, sediments on the Canadian side of the river have been systematically re-sampled, but the US side of the river has received less attention. This paper discusses two recent projects, one completed in 2003 and one completed in 2005, that sought to revisit a variety of sediment localities within the St. Marys River to update sediment contaminant data, and to expand sampling into additional areas of interest on the US side of the border: Munuscong Lake at the southern end of the AOC, and the Little Rapids adjacent to Sugar Island. The primary focus was on surficial sediment contamination, particularly metals shown to be elevated during previous sampling (chromium, nickel, copper). Results show that while Cr, Ni and Cu contamination remains slightly elevated in surficial sediments at some sites in the St. Marys River, the overall trend shows metal concentrations declining to levels consistent with background levels in sediments found in Lake Superior, Whitefish Bay and the St. Marys River.

Early mortality syndrome (EMS) is the term used to describe the mortality in early life stages of salmonids due to thiamine deficiency within the Great Lakes basin. Since 1985, Lake Superior State University (LSSU) has raised Atlantic salmon Salmo salar for stocking in the St. Marys River region near Sault Ste. Marie, Michigan. In 2000, the Atlantic salmon raised at LSSU experienced 99% mortality due to EMS related symptoms. In order to better understand and document the incidence of EMS in the St. Marys River population of Atlantic salmon, egg-thiamine content has been measured in this population since 2003. Egg-thiamine levels were measured using a rapid reversed-phase solid-phase extraction (RP–SPE) method of thiamine analysis. This paper presents the egg-thiamine data for this population of Atlantic salmon for the years 2003–2007. Over this five-year period, the egg-thiamine content of eggs from 2005 saw a significant increase. This one year shift may have larger ecological implications related to changes in the diet of this population of Atlantic salmon.

Early mortality syndrome (EMS) is the term used to describe the mortality of juvenile salmonids due to thiamine deficiency within the Great Lakes basin. EMS appears to be related to the presence of thiaminase found in prey fish such as alewives Alosa pseudoharengus, rainbow smelt Osmerus mordax, and gizzard shad Dorosoma cepedianum. Since 1985, the Michigan Department of Natural Resources, in conjunction with Lake Superior State University's Aquatic Research Laboratory, has raised Atlantic salmon Salmo salar for stocking in the St. Marys River region near Sault Ste. Marie, Michigan. The current study correlates egg-thiamine concentrations from 45 individual family groups with egg and larval mortality as well as length, weight, and condition data obtained from spawning females. Thiamine concentrations were measured at three stages of egg-development using a newly developed rapid reversed-phase solid-phase extraction (RP-SPE) method of thiamine analysis. Results suggest a threshold average total egg-thiamine concentration of approximately 1.0 (nmol/g egg) for normal larval survival. Thiamine analysis indicated no significant change in egg-thiamine concentrations from prefertilization to the eyed stage of development. Female parent weight showed an inverse relationship with egg-thiamine levels. Thiamine immersion of larval sac-fry eliminated the occurrence of EMS for this study population. Due to low cost and ease of use, the RP-SPE method for large scale egg-thiamine analysis has the potential to impact basin wide management decisions with respect to salmonid stocking programs.

The DNA repair enzyme uracil DNA glycosylase catalyzes the first step in the uracil base excision repair pathway, the hydrolytic cleavage of the N-glycosidic bond of deoxyuridine in DNA. Here we report kinetic isotope effect (KIE) measurements that have allowed the determination of the transition-state structure for this important reaction. The small primary (13)C KIE (=1.010 +/- 0.009) and the large secondary alpha-deuterium KIE (=1.201 +/- 0.021) indicate that (i) the glycosidic bond is essentially completely broken in the transition state and (ii) there is significant sp(2) character at the anomeric carbon. Large secondary beta-deuterium KIEs were observed when [2'R-(2)H] = 1.102 +/- 0.011 and [2'S-(2)H] = 1.106 +/- 0.010. The nearly equal and large magnitudes of the two stereospecific beta-deuterium KIEs indicate strong hyperconjugation between the elongated glycosidic bond and both of the C2'-H2' bonds. Geometric interpretation of these beta-deuterium KIEs indicates that the furanose ring adopts a mild 3'-exo sugar pucker in the transition state, as would be expected for maximal stabilization of an oxocarbenium ion. Taken together, these results strongly indicate that the reaction proceeds through a dissociative transition state, with complete dissociation of the uracil anion followed by addition of water. To our knowledge, this is the first transition-state structure determined for enzymatic cleavage of the glycosidic linkage in a pyrimidine deoxyribonucleotide.

The DNA repair enzyme uracil DNA glycosylase (UDG) pinches the phosphodiester backbone of damaged DNA using the hydroxyl side chains of a conserved trio of serine residues, resulting in flipping of the deoxyuridine from the DNA helix into the enzyme active site. We have investigated the energetic role of these serine-phosphodiester interactions using the complementary approaches of crystallography, directed mutagenesis, and stereospecific phosphorothioate substitutions. A new crystal structure of UDG bound to 5'-HO-dUAAp-3' (which lacks the 5' phosphodiester group that interacts with the Ser88 pinching finger) shows that the glycosidic bond of dU has been cleaved, and that the enzyme has undergone the same specific clamping motion that brings key active site groups into position as previously observed in the structures of human UDG bound to large duplex DNA substrates. From this structure, it may be concluded that glycosidic bond cleavage and the induced fit conformational change in UDG can occur without the 5' pinching interaction. The S88A, S189A, and S192G "pinching" mutations exhibit 360-, 80-, and 21-fold damaging effects on k(cat)/K(m), respectively, while the S88A/S189A double mutant exhibits an 8200-fold damaging effect. A free energy analysis of the combined effects of nonbridging phosphorothioate substitution and mutation at these positions reveals the presence of a modest amount of strain energy between the compressed 5' and 3' phosphodiester groups flanking the bound uridine. Overall, these results indicate a role for these serine-phosphodiester interactions in uracil flipping and preorganization of the sugar ring into a reactive conformation. However, in contrast to a recent proposal [Parikh, S. S., et al. (2000) Proc Natl. Acad. Sci. 94, 5083], there is no evidence that conformational strain of the glycosidic bond induced by serine pinching plays a major role in the 10(12)-fold rate enhancement brought about by UDG.

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The amino acids 5-diazo-4-oxo-L-norvaline, 4-oxo-L-norvaline, and (methanesulfinyl)-L-alanine have been incorporated into three separate tripeptides wherein these nonnatural amino acids replace Asn in a known tripeptide substrate of oligosaccharyltransferase. Synthesis of both the diazoketone- and sulfoxide-containing peptides involved functionalization of the appropriate side chain after peptide assembly, whereas synthesis of the methyl ketone-containing peptide was effected by synthesis of the protected amino acid followed by its incorporation into the desired tripeptide. None of the three synthetic tripeptides showed activity as substrates, nor were they potent inhibitors of oligosaccharyltransferase at concentrations that were 10-35 times the K-m for the corresponding Asn-containing tripeptide substrate. NMR analysis in DMSO-d(6) showed that the diazoketone and methyl ketone peptides adopt the "Asx-turn" conformation, which has been postulated to be crucial for substrate binding. Furthermore, a nonsubstrate peptide, Ac-Asn-Pro-Thr-NH2, was found to adopt the "Asx-turn" in both the solid state and in solution(DMSO-d(6)). The collective data suggest that the ability to form an Asx-turn in the N-glycosylation consensus sequence (Asn-Xaa-Ser/Thr) may be a necessary but not sufficient condition for substrate binding and catalysis.

The synthesis of a new glycoamino acid derivative, a direct C-analog of N-4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-Asn is described. The C-glycoside is prepared by a tandem Horner-Emmons-Wadsworth olefination-Michael addition between an aspartyl beta-keto phosphonate and a 4,6-O-benzylidene GlcNAc sugar. (C) 1998 Elsevier Science Ltd. All rights reserved.

ADVERTISEMENT RETURN TO ISSUEPREVNoteNEXTPreparation of 4-Oxo-l-norvaline via Diazomethane Homologation of β-Aspartyl SemialdehydeR. Marshall Werner, Ori Shokek, and Jeffery T. DavisView Author Information Department of Chemistry and Biochemistry, University of Maryland at College Park, College Park, Maryland 20742 Cite this: J. Org. Chem. 1997, 62, 23, 8243–8246Publication Date (Web):November 14, 1997Publication History Received16 June 1997Published online14 November 1997Published inissue 1 November 1997https://pubs.acs.org/doi/10.1021/jo971088ahttps://doi.org/10.1021/jo971088abrief-reportACS PublicationsCopyright © 1997 American Chemical SocietyRequest reuse permissionsArticle Views840Altmetric-Citations30LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-AlertscloseSupporting Info (1)»Supporting Information Supporting Information SUBJECTS:Aldehydes,Inhibitors,Ketones,Peptides and proteins,Protective groups Get e-Alerts