Sequence-specific double-strand cleavage of DNA by penta-N-methylpyrrolecarboxamide-EDTA·Fe(II)

In the presence of O2 and 5 mM dithiothreitol, penta-N-methylpyrrolecarboxamide-EDTA·Fe(II) [P5E·Fe(II)] at 0.5 µ M cleaves pBR322 plasmid DNA (50 µ M in base pairs) on opposite strands to afford discrete DNA fragments as analyzed by agarose gel electrophoresis. High-resolution denaturing gel electrophoresis of a 32P-end-labeled 517-base-pair restriction fragment containing a major cleavage site reveals that P5E·Fe(II) cleaves 3-5 base pairs contiguous to a 6-base-pair sequence, 5'-T-T-T-T-T-A-3' (4,323-4,328 base pairs). The major binding orientation of the pentapeptide occurs with the amino terminus at the adenine side of this sequence. In the presence of 5 mM dithiothreitol, 0.01 µ M P5E·Fe(II) converts form I pBR322 DNA at 0.22 µ M plasmid (1.0 mM in base pairs) to 40% form II, indicating the cleavage reaction is catalytic, turning over a minimum of nine times. This synthetic molecule achieves double-strand cleavage of DNA (pH 7.9, 25 degrees C) at the 6-base-pair recognition level and may provide an approach to the design of "artificial restriction enzymes.

Insights into antibody catalysis: Structure of an oxygenation catalyst at 1.9-Å resolution

The x-ray crystal structures of the sulfide oxidase antibody 28B4 and of antibody 28B4 complexed with hapten have been solved at 2.2-Å and 1.9-Å resolution, respectively. To our knowledge, these structures are the highest resolution catalytic antibody structures to date and provide insight into the molecular mechanism of this antibody-catalyzed monooxygenation reaction. Specifically, the data suggest that entropic restriction plays a fundamental role in catalysis through the precise alignment of the thioether substrate and oxidant. The antibody active site also stabilizes developing charge on both sulfur and periodate in the transition state via cation-pi and electrostatic interactions, respectively. In addition to demonstrating that the active site of antibody 28B4 does indeed reflect the mechanistic information programmed in the aminophosphonic acid hapten, these high-resolution structures provide a basis for enhancing turnover rates through mutagenesis and improved hapten design

Are Men Cheaper Than Women? Insights From Walking Economy

ARE MEN CHEAPER THAN WOMEN? INSIGHTS FROM WALKING ECONOMY Nicole S. Schultz and Peter G. Weyand Southern Methodist University, Locomotor Performance Laboratory, Department of Applied Physiology and Wellness, Dallas, TX 75205 Introduction: The metabolic energy cost of human walking has been extensively studied. However, whether men and women require the same amount of energy (per kg of body mass) to walk at the same speed or cover the same distance remains unresolved. While most predictive algorithms incorporate only body mass and walking speed, we have recently found that mass-specific walking metabolic rates are inversely related to stature. Objective: We tested the specific hypothesis that walking economy does not differ between males and females when stature is controlled. We evaluated our hypothesis by comparing stature-matched adult males vs. females in each of three categories: short, medium and tall, at their respective most economical walking speeds. We predicted that minimum transport costs (COTmin, ml O2·kg-1·m-1) would not vary between gender groups of each stature. Methods: 30 subjects (15 male, 15 female) walked on a level treadmill at speeds ranging from 0.4 m·s-1 to 1.9 m·s-1. Indirect calorimetry was used to measure rates of oxygen uptake under steady-state conditions (Parvo Medics TrueOne 2400, Sandy, UT). Analysis: Minimum net transport cost, or net transport cost at the most economical walking speed, was determined for each subject by: 1) subtracting resting rates of oxygen uptake from the gross rates measured (VO2net, ml O2·kg-1·min-1), and 2) dividing VO2net by walking speed to identify the minimum COT. Unpaired t-tests were used to compare males and females within each stature group. Results: Transport costs were essentially identical for male and female groups of all three statures: short, medium and tall, and were inversely related to stature as expected. Conclusion: We conclude that men and women are equally cheap and gender does not influence walking energy expenditure. The mass-specific energy cost of transport does not differ in males and females of the same stature

An Expanded Genetic Code In Mammalian Cells With A Functional Quadruplet Codon

We have utilized in vitro evolution to identify tRNA variants with significantly enhanced activity for the incorporation of unnatural amino acids into proteins in response to a quadruplet codon in both bacterial and mammalian cells. This approach will facilitate the creation of an optimized and standardized system for the genetic incorporation of unnatural amino acids using quadruplet codons, which will allow the biosynthesis of biopolymers that contain multiple unnatural building blocks

Photocleavage of the Polypeptide Backbone by 2-Nitrophenylalanine

SummaryPhotocleavage of the polypeptide backbone is potentially a powerful and general method to activate or deactivate functional peptides and proteins with high spatial and temporal resolution. Here we show that 2-nitrophenylalanine is able to photochemically cleave the polypeptide backbone by an unusual cinnoline-forming reaction. This unnatural amino acid was genetically encoded in E. coli, and protein containing 2-nitrophenylalanine was expressed and site-specifically photocleaved

Slowing Down of Positrons and Applications to Solid Surfaces

When monoenergetic positrons enter a solid they scatter and lose energy via processes similar to those for electrons. Theoretical details of these processes have been well established for decades, but experimental results using low energy positron beams are only now becoming available for comparison. We review the theoretical results for elastic and inelastic scattering of positrons and the predictions that follow for backscattering, inner-shell ionization, energy loss and stopping profiles. In this presentation, emphasis is given to specific comparisons with calculations for electrons. We discuss recent experimental results in each of these areas, and conclude with two examples of applications of positron beam techniques to near-surface research