The Structure of Maltooctaose-Bound Escherichia coli Branching Enzyme Suggests a Mechanism for Donor Chain Specificity.

Glycogen is the primary storage polysaccharide in bacteria and animals. It is a glucose polymer linked by α-1,4 glucose linkages and branched via α-1,6-linkages, with the latter reaction catalyzed by branching enzymes. Both the length and dispensation of these branches are critical in defining the structure, density, and relative bioavailability of the storage polysaccharide. Key to this is the specificity of branching enzymes because they define branch length. Herein, we report the crystal structure of the maltooctaose-bound branching enzyme from the enterobacteria E. coli. The structure identifies three new malto-oligosaccharide binding sites and confirms oligosaccharide binding in seven others, bringing the total number of oligosaccharide binding sites to twelve. In addition, the structure shows distinctly different binding in previously identified site I, with a substantially longer glucan chain ordered in the binding site. Using the donor oligosaccharide chain-bound Cyanothece branching enzyme structure as a guide, binding site I was identified as the likely binding surface for the extended donor chains that the E. coli branching enzyme is known to transfer. Furthermore, the structure suggests that analogous loops in branching enzymes from a diversity of organisms are responsible for branch chain length specificity. Together, these results suggest a possible mechanism for transfer chain specificity involving some of these surface binding sites.

Reveal Q+ MAX® for Detection of Total Af latoxin in Corn, Almonds, Pistachios, Walnuts, and Peanuts.

Background: The Reveal Q+ MAX for Aflatoxin is a lateral flow immunochromatographic test intended for quantitative analysis within 6 min after aqueous extraction. Objective: Work was conducted to validate the performance of the Reveal Q+ MAX for Aflatoxin method in selected corn and nut matrixes. Methods: This method was validated under the requirements of the AOAC Research Institute Performance Tested MethodSM program. Five matrixes, including corn naturally contaminated with aflatoxin at 0, 5.2, 21.0, 51.6, 103.6, and 282 ppb as well as peanuts, pistachios, walnuts, and almonds spiked at 0, 5, 20, 50, and 300 ppb were analyzed. Results: Average percentage recoveries of the added aflatoxin from the matrixes ranged from 80.8 to 116.9%. Average LOD for all matrixes is 2 ppb and LOQ is 7 ppb. With the exception of sample size for almonds, robustness trials demonstrated that deliberate changes to the assay parameters minimally affected the Reveal Q+ MAX assay performance. Finally, stability results from three independently manufactured lots support Reveal Q+ MAX for Aflatoxin performance consistency and shelf-life of 18 months when stored at room temperature. Conclusions: This study appropriately validates the Performance Tested MethodSM claim for corn and selected nut matrixes on Reveal Q+ MAX for Aflatoxin, an aqueous lateral flow test kit. Highlights: Aqueous lateral flow test kit detects total aflatoxin between 80 to 120% yield with an LOD of 2 ppb.

Crystal Structures of Escherichia coli Branching Enzyme in Complex with Linear Oligosaccharides.

Branching enzyme is responsible for all branching of glycogen and starch. It is an unusual member of the α-amylase family because it has both α-1,4-amylase activity and α-1,6-transferase activity [Drummond, G. S., et al. (1972) Eur. J. Biochem. 26, 168-176]. It also does not react with shorter glucans, though it will bind much longer substrates and substrate mimics [Binderup, K., et al. (2002) Arch. Biochem. Biophys. 397, 279-285]. In an effort to better understand how branching enzyme interacts with its polymeric substrate, we have determined the structure of Δ112 Escherichia coli branching enzyme bound to maltoheptaose and maltohexaose. Together, these structures define six distinct oligosaccharide binding sites on the surface of E. coli branching enzyme. Most of these binding sites surround the edge of the ß-barrel domain and are quite far from the active site. Surprisingly, there is no evidence of oligosaccharide binding in the active site of the enzyme. The closest bound oligosaccharide resides almost 18 Å from the active site. Mutations to conserved residues in binding sites I and VI had a debilitating effect on the activity of the enzyme.

Improvements in self-administration studies based on changes in skin button type and surgical technique.

INTRODUCTION: These studies, ranging in duration from 3 to 8months, evaluated the patency and longevity of the intravenous (IV) self-administration surgical model in male Sprague Dawley rats. Surgeries were categorized and assessed based on the number of catheter and/or skin button repairs required per animal across four separate self-administration studies. Design improvements in skin button types and changes in surgical procedures were chronologically tracked and assessed. METHODS: Animals were evaluated under a self-administration paradigm in which they were trained to respond for a food reward under a fixed ratio schedule (FR5 or FR10). Animals were then surgically prepared with a femoral catheter and skin button port. Following recovery, animals were returned to food-maintained responding for at least 5 sessions and subsequently trained to respond for injections of a reinforcing drug. Once drug training criteria was established, the effects of vehicle or varying doses of test articles were evaluated. Animals were tested in operant chambers one hour each day 5days a week and the length of each study was recorded. Differences in the number of repairs per study as well as the total number of repairs were tabulated. RESULTS: Study length was directly correlated to the mean number of repairs occurring per study, with study length increasing as the total number of repairs increased. The majority of repairs were skin button-related issues. Multiple combinations of skin button types and surgical techniques were implemented across time to evaluate model efficiency and decrease overall cycle time per study. Initial combinations produced a greater number of repairs on a per study basis. However, the skin button type and surgical technique combination that resulted in the fewest number of total repairs used a lateral incision with a dorsal biopsy punch. DISCUSSION: The combination of improvements in skin button type and surgical techniques drastically decreased the number of surgical repairs required per study, increasing efficiency and thereby decreasing the overall cycle time for study completion.

A behavioral economic analysis of the effect of next-day responsibilities on drinking.

Approximately 37% of college students report heavy episodic drinking (5 or more drinks in an occasion for men and 4 or more for women) in the past month. This pattern of drinking is often associated with high blood alcohol levels, accidents, injuries, and negative social and academic outcomes. There is a need for novel theoretical approaches to guide prevention efforts. Behavioral economics emphasizes the role of contextual determinants, such as drink price and the presence and amount of alternative reinforcement as determinants of drinking levels and has received strong empirical support in basic laboratory research. This translational research study used a hypothetical behavioral economic measure to investigate the impact of a variety of next-day responsibilities on night-before drinking intentions in a sample of first-year college students (N = 80; 50% female) who reported recent heavy episodic drinking. Drinking estimates were significantly lower in all of the responsibility conditions relative to the no-responsibility condition; internships were associated with the greatest reduction (d(rm) = 1.72), and earlier class times were associated with greater reductions in drinking intentions (d(rm) range = 1.22-1.35) than later class times (d(rm) range = 0.83-1.00). These results suggest that increasing morning responsibilities should be further investigated as a potential strategy to reduce drinking in college students.