Helicase activity on DNA as a propagating front

We develop a propagating front analysis, in terms of a local probability of zipping, for the helicase activity of opening up a double stranded DNA (dsDNA). In a fixed-distance ensemble (conjugate to the fixed-force ensemble) the front separates the zipped and unzipped phases of a dsDNA and a drive acts locally around the front. Bounds from variational analysis and numerical estimates for the speed of a helicase are obtained. Different types of helicase behaviours can be distinguished by the nature of the drive.Comment: 5 pages, 5 eps figures; replaced by the published versio

Positive Youth Development Life Skills Gained at the Iowa 4-H Youth Conference

Research suggests 4-H programs build Life Skills such as leadership, communication, citizenship and learning. However, 4-H programs vary from long-term on-going experiences to shorter, more intense opportunities. This paper discusses a program evaluation articulating the life skill development of participants in a 3-day residential State 4-H Conference on a Midwestern college campus. The Life Skills assessed were in the areas of leadership, citizenship, communication, and learning as part of overall Life Skill development. Participants were youth ages 14-18 years. A retrospective pretest-posttest was used to evaluate skill development and understanding. Analysis, including paired sample t-tests, indicated growth in each of the 12 common outcome measures evaluated. This study supports the importance of purposeful planning and youth engagement in the learning process to achieve desired life skill outcomes

Multiple C-Terminal Tails within a Single \u3cem\u3eE. coli\u3c/em\u3e SSB Homotetramer Coordinate DNA Replication and Repair

Escherichia coli single-stranded DNA binding protein (SSB) plays essential roles in DNA replication, recombination and repair. SSB functions as a homotetramer with each subunit possessing a DNA binding domain (OB-fold) and an intrinsically disordered C-terminus, of which the last nine amino acids provide the site for interaction with at least a dozen other proteins that function in DNA metabolism. To examine how many C-termini are needed for SSB function, we engineered covalently linked forms of SSB that possess only one or two C-termini within a four-OB-fold “tetramer”. Whereas E. coli expressing SSB with only two tails can survive, expression of a single-tailed SSB is dominant lethal. E. coli expressing only the two-tailed SSB recovers faster from exposure to DNA damaging agents but accumulates more mutations. A single-tailed SSB shows defects in coupled leading and lagging strand DNA replication and does not support replication restart in vitro. These deficiencies in vitro provide a plausible explanation for the lethality observed in vivo. These results indicate that a single SSB tetramer must interact simultaneously with multiple protein partners during some essential roles in genome maintenance

Velocity and processivity of helicase unwinding of double-stranded nucleic acids

Helicases are molecular motors which unwind double-stranded nucleic acids (dsNA) in cells. Many helicases move with directional bias on single-stranded (ss) nucleic acids, and couple their directional translocation to strand separation. A model of the coupling between translocation and unwinding uses an interaction potential to represent passive and active helicase mechanisms. A passive helicase must wait for thermal fluctuations to open dsNA base pairs before it can advance and inhibit NA closing. An active helicase directly destabilizes dsNA base pairs, accelerating the opening rate. Here we extend this model to include helicase unbinding from the nucleic-acid strand. The helicase processivity depends on the form of the interaction potential. A passive helicase has a mean attachment time which does not change between ss translocation and ds unwinding, while an active helicase in general shows a decrease in attachment time during unwinding relative to ss translocation. In addition, we describe how helicase unwinding velocity and processivity vary if the base-pair binding free energy is changed.Comment: To appear in special issue on molecular motors, Journal of Physics - Condensed Matte

Effect of Tai Chi Exercise Combined with Mental Imagery Theory in Improving Balance in a Diabetic and Elderly Population

BACKGROUND: One of the effects of diabetes mellitus (DM), peripheral neuropathy, affects the sensation in the feet and can increase the chance of falling. The purpose of the study was to investigate the effect of 8 weeks of Tai Chi (TC) training combined with mental imagery (MI) on improving balance in people with diabetes and an age matched control group.MATERIAL AND METHODS: Seventeen healthy subjects and 12 diabetic sedentary subjects ranging from 40–80 years of age were recruited. All subjects in both groups attended a Yang style of TC class using MI strategies, 2 sessions a week for 8 weeks. Each session was one hour long. Measures were taken using a balance platform test, an Activities-specific Balance Confidence (ABC) Scale, a one leg standing test (OLS), functional reach test (FRT) and hemoglobin A1C. These measures were taken twice, pre and post-study, for both groups.RESULTS: Both groups experienced significant improvements in ABC, OLS, FRT (P\u3c0.01) after completing 8 weeks of TC exercise with no significant improvement between groups. Subjects using the balance platform test demonstrated improvement in balance in all different tasks with no significant change between groups. There was no significant change in HbA1C for the diabetic group.CONCLUSIONS: All results showed an improvement in balance in the diabetic and the control groups; however, no significant difference between the groups was observed. Since the DM group had more problems with balance impairment at baseline than the control, the diabetic group showed the most benefit from the TC exercise

Engineering a reagentless biosensor for single-stranded DNA to measure real-time helicase activity in Bacillus

Single-stranded DNA-binding protein(SSB)is a well characterized ubiquitous and essential bacterial protein involved in almost all aspects of DNA metabolism. Using the Bacillus subtilis SSB we have generated areagentless SSB biosensor that can be used as a helicase probe in B. subtilis and closely related gram positive bacteria. We have demonstrated the utility of the probe in a DNA unwinding reaction using a helicase from Bacillus and for the first time,characterized the B. subtilis SSB's DNA binding mode switching and stoichiometry.The importance of SSB in DNA metabolism is not limited to simply binding and protecting ssDNA during DNA replication, as previously thought. It interacts with an array of partner proteins to coordinate many different aspects of DNA metabolism. In most cases its interactions with partner proteins is species-specific and for this reason, knowing how to produce and use cognate reagentless SSB biosensors indifferent bacteria is critical.Here we explain how to produce a B. subtilis SSB probe that exhibits 9-fold fluorescence increase upon binding to single stranded DNA and can be used in all related grampositive firmicutes which employ drastically different DNA replication and repair systems than the widely studied Escherichiacoli. The materials to produce the B. subtilis SSB probe a recommercially available, so the methodology described here is widely available unlike previously published methods for the E. coli SSB

Six Weeks Habituation of Simulated Barefoot Running Induces Neuromuscular Adaptations and Changes in Foot Strike Patterns in Female Runners

BACKGROUND: The aim of this study was to examine the effects of a 6-week training program of simulated barefoot running (SBR) on running kinetics in habitually shod (wearing shoes) female recreational runners.MATERIAL AND METHODS: Twelve female runners age 25.7±3.4 years gradually increased running distance in Vibram FiveFingers minimal shoes over a 6-week period. The kinetic analysis of treadmill running at 10 Km/h was performed pre- and post-intervention in shod running, non-habituated SBR, and habituated SBR conditions. Spatiotemporal parameters, ground reaction force components, and electromyography (EMG) were measured in all conditions.RESULTS: Post-intervention data indicated a significant decrease across time in the habituation SBR for EMG activity of the tibialis anterior (TA) in the pre-activation and absorptive phase of running (PCONCLUSIONS: The findings of this study indicate that changes in motor patterns in previously habitually shod runners are possible and can be accomplished within 6 weeks. Non-habituation SBR did not show a significant neuromuscular adaptation in the EMG activity of TA and GAS as manifested after 6 weeks of habituated SBR

The Effect of a Femoral Fracture Sustained before Skeletal Maturity on Bone Mineral Density: A Long-Term Follow-Up Study

Peer reviewe

Efforts towards the synthesis of fully N-differentiated heparin-like glycosaminoglycans; and, Investigations into the mechanism of inactivation of RTPR by gemcitabine triphosphate

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, February 2007.Vita.Includes bibliographical references.Efforts towards the Synthesis of Fully N-Differentiated Heparin-like Glycosaminoglycans. Heparin-like glycosaminoglycans (HLGAGs) are complex information-carrying biopolymers and are an important component of the coagulation cascade. They have also been implicated in interactions with growth factors, cytokines, virus entry, and other functions. Currently, no general synthesis of arbitrary HLGAG sequences has been demonstrated. The modular synthesis of glycosaminoglycans requires straightforward methods for the production of large quantities of protected uronic acid building blocks. An efficient route to methyl 3-0- benzyl-1,2-O-isopropylidene-a-L-idopyranosiduronate from diacetone glucose in nine steps and 36% overall yield is described. Additionally, a general method for the conversion of glycals to the corresponding 1,2-cis-isopropylidene-a-glycosides is reported. Epoxidation of glycals with dimethyldioxirane followed by ZnC12-catalyzed addition of acetone converted a variety of protected glycals into 1,2-cis-isopropylidene-a-glycosides in good yield. The reaction is compatible with a range of protecting groups, as well as free hydroxyl groups. This method has been applied to develop a synthesis of 3-O-benzyl-1,2-O-isopropylidene-P-D-glucopyranosiduronate in seven steps and 32% overall yield.(cont.) These compounds are useful as glycosyl acceptors and as intermediates that may be further elaborated into uronic acid trichloroacetimidate glycosyl donors for the assembly of glycosaminoglycan structures. The glucosamine residues in HLGAGs have been found to exist as amines, acetamides, and N-sulfonates. In order to develop a completely general, modular synthesis of heparin, three degrees of orthogonal nitrogen protection are required. Reported is a strategy for the synthesis of fully N-differentiated heparin oligosaccharides in the context of target octasaccharide 3-1, which contains an N-acetate, N-sulfonates, and a free amine. The protecting group scheme used in the synthesis blocked the N-acetate as a N-diacetate, the N-sulfonates as azido groups, and the amine as a N-CBz; free hydroxyls were masked as benzyl ethers and O-sulfonates as acetate esters. Disaccharide and tetrasaccharide modules were synthesized using this strategy; however, the union of tetrasaccharide trichloroacetimidate 3-4 with disaccharide acceptor 3-5 unexpectedly formed the undesired P-linked glycoside in addition to the a-linkage anticipated for iduronic acid nucleophiles, resulting in an inseparable 6:1 a: p mixture of products. Detailed studies into the basis for this unexpected result were conducted and are also reported.(cont.) Investigations into the Mechanism of Inactivation of RTPR by Gemcitabine Triphosphate. Ribonucleoside triphosphate reductase (RTPR) is an adenosylcobalamin (AdoCbl) dependant enzyme that catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates via controlled radical chemistry. The antitumor agent 2',2'-difluoro-2'- deoxycytidine (gemcitabine, F2C) has been shown to owe some of its in vivo activity to inhibition of human RNR by the 5'-diphosphate (F2CDP). Previous studies have shown that RTPR is rapidly inactivated by one equivalent of 2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (F2CTP). This inactivation is associated with the release of two equivalents of fluoride and modification of RTPR by a Co-S bond between C419 and the cobalamin cofactor. In order to further characterize this inactivation, isotopically labeled derivatives of F2CTP were synthesized: radiolabeled 1'-[3H]-F2C and mass labeled 1'-[2H]-F2C and 3'-[2H]-F2C. These compounds were converted to F2CTP through a set of enzymatic phosphorylation steps which overcome difficulties found using traditional, chemical methods. Biochemical investigations were performed using these labeled derivatives to track the fate of the base and sugar during RTPR inactivation by F2CTP.(cont.) The release of cytosine base, previously overlooked in this system, was detected utilizing 5-[3H]-F2CTP: 0.7 equiv. of cytosine were released, with 0.15-0.2 equiv. of unreacted F2CTP remaining. Size exclusion chromatography (SEC) was used to quantify covalent labeling of RTPR by F2CTP: 0.15 equiv. were detected using 5-[3H]-F2CTP, 0.45 equiv. were detected using 1'-[3H]-F2CTP. A small molecule nucleotide product was identified in inactivation mixtures quenched with NaBH4 and identified as an isomer of cytidine, indicating the loss of both fluorides and the addition of an oxygen at the 2' carbon. RTPR inactivated with 1'-[3H]-F2CTP was digested with trypsin and peptides containing radioactivity purified. Identical peptides were prepared using partially deuterated F2CTP, allowing identification by MALDI-MS. Post source decay (PSD) MS/MS methods were used to further characterize these peptides, identifying the site of label as the C-terminal tryptic peptide of RTPR at C731 and C736. The cysteines were labeled through conjugate addition with a furanone-like precursor that had lost cytosine, triphosphate, and both fluorines. The results of these studies have allowed for the first time the proposal of a mechanistic hypothesis for RTPR inactivation by F2CTP.by Gregory J.S. Lohman.Ph.D

17β-Estradiol Induced Effects on Anterior Cruciate Ligament Laxness and Neuromuscular Activation Patterns in Female Runners

Background: To investigate the effects of 17β-Estradiol across phases of menstrual cycle on the laxness of the anterior cruciate ligament (ACL) and the neuromuscular control around the knee joint in female runners. Methods: Twelve healthy female runners, who reported normal menstrual cycles for the previous 6 months were tested twice across one complete menstrual cycle for serum levels of 17β Estradiol (E), and knee joint laxity (KJL). Electromyographic (EMG) activity of the quadriceps and hamstrings muscles was also recorded during running on a treadmill. The changes in the EMG activity, KJL, and hormonal concentrations were recorded for each subject during the follicular and the ovulatory phases across the menstrual cycle. Results: An observed increased in KJL in response to peak E during the ovulatory phase, was associated with increased preactivity of the hamstring muscle before foot impact (p \u3c 0.001). A consistent pattern was also observed in the firing of the quadriceps muscle recruitment pattern throughout the follicular phase associated with decreased hamstring recruitment pattern during weight acceptance phase of running (p = 0.02). Additionally, low ratio of medial to lateral quadriceps recruitment was associated with a significant reduction of the quadriceps to hamstring cocontraction ratio during the follicular phase. Conclusions: Changes in KJL during the menstrual cycle in response to 17β-Estradiol fluctuations changes the neuromuscular control around the knee during running. Female runners utilize different neuromuscular control strategies during different phases of the menstrual cycle which may contribute to increase ACL injury risk