Investigations of the PPi-dependent Acetate Kinase from the Parasite \u3ci\u3eEntamoeba histolytica\u3c/i\u3e

Acetate, a short-chain fatty acid that plays a key role in all domains of life, can be utilized as a carbon source or excreted as a product of metabolism. Acetate kinase (ACK), a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as the phosphoryl donor. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya. A partially purified ACK which can utilize pyrophosphate (PPi) as the phosphoryl donor in the acetyl phosphate-forming direction was previously identified in Entamoeba histolytica, the causative agent of amoebic dysentery. Here I describe my research on the E. histolytica PPi-dependent ACK. My biochemical and kinetic characterization of the E. histolytica ACK revealed several properties that are unique to this enzyme. First, the E. histolytica enzyme is capable of utilizing acyl substrates longer than butyrate and can utilize acyl substrates as long as hexanoate in the direction of acetyl phosphate synthesis. Second, this ACK can only utilize PPi as a phosphoryl donor and cannot utilize ATP. The kinetic parameters for substrate affinity indicate that the enzyme prefers both an acetate and propionate in the acetyl phosphate-forming direction; however, the low kcat raises question whether this direction of the reaction is physiological. The enzyme operates preferentially in the direction of acetate/PPisynthesis as the rate of catalysis and overall catalytic efficiency are 1,102-fold and 240,351-fold higher, respectively. Studies of the mechanism of this enzyme indicates that the PPi-ACK follows a sequential mechanism, supporting a direct in-line phosphoryl transfer mechanism previously reported in the well characterized Methanosarcina thermophila ATP-dependent ACK. A modified hydroxamate assay was developed for measuring ACKs in the direction of acetate formation. A coupled enzyme assay has been used to measure ATP production in the direction for the prokaryotic ACKs, but there was not a method available for measuring a PPi-forming ACK in this direction. The assay described here allowed for the determination of kinetic parameters in the acetate-forming direction for the E. histolytica ACK and can not only be used to measure ACK activity of both ATP-dependent and PPi-dependent acetate kinases in the direction of acetate formation, but could also prove useful for other enzymes that utilize activated acyl substrates such as acetyl adenylate and acetyl-CoA. Investigation of the residues in the putative PPi-ACK active site in E. histolytica revealed that while residues conserved across the ACK family are present in the active site, the enzyme has a number of significant active site changes in comparison to its counterpart, the M. thermophila ATP-dependent ACK. The residues His117, His172, Val87, Thr201, Thr15, Arg274, and Asp272 appear to be essential for catalysis in the direction of acetate synthesis. Furthermore, His117 appears to be critically important for binding acetyl phosphate and therefore important in acetate/PPi synthesis reactions. RNA interference of E. histolytica ACK was unable to be carried out due to problems with transfection of plasmid DNA into the parasite. However, data garnered from cellular extracts indicates that E. histolytica has a PPi-dependent ACK but not ATP-dependent ACK. Activities of the eukaryotic ACK partner enzymes phosphotransacetylase and xylulose 5-phosphate/fructose 6- phosphate phosphoketolase were absent in cellular extracts. The absence of bacterial ACK partner enzymes in Entamoeba suggests that a novel acetyl phosphate generating enzyme is the partner for the PPi-ACK in this protist. This speculative partner enzyme could generate acetyl phosphate from an intermediate of the pentose phosphate pathway or the end product of a yet to be identified catabolic pathway

Threat, violence, and voters: race and context in the 2008 presidential election

The 2008 Presidential Election brought into office the first African-American president in U.S. history. This paper analyzes variations in White support for Barack Obama based on a number of county-level contextual factors, which are hypothesized to influence aggregate White voter support for the Democratic candidate. Based on the well-known racial threat theory, this paper will explore how racial composition and income inequality effect White support for Barack Obama. Another key explanatory variable, violence, is thought to influence White voter support because of the preconceptions some of these voters hold about African-Americans. Violence helps shape the stereotypes White voters hold, and these stereotypes are not left behind when entering the voting booth. If violence helps explain variation in county-level White support for Barack Obama, this paper offers preliminary evidence that stereotypes about violence may have a significant influence on voting and African-American candidate strategies may not have the sway necessary to overcome some of these barriers in the electoral arena

Chinese Public Education and Global Social Justice: The Organizational Challenges of Initiating an Access Scholarship in a Chinese Public School

This OIP aims to support ABC Academy in its efforts to enable Middle Eastern refugees and other economically and politically marginalized youth to complete their final year of high school in Beijing, China. Transformative leadership, grounded in notions of social justice and democracy, provide a guide for an institution that seeks to bridge the divide between classroom education and social context. A robust framework for implementing change within an insular organization is proposed wherein transformative leadership’s key tenets are augmented by Kotter’s eight-step change model (1996). Capacity building and empowerment provide faculty with the tools necessary to improve student learning and postsecondary preparedness. Institutional leaders can create lasting change by cultivating an environment with shared purpose, frequent dialogue, and detailed frameworks for assessing change effectiveness. The change plan hopes to create holistic learning opportunities in the Chinese education system that aid local and foreign students, leading to improvements within the classroom and beyond it

Creating a Livestock Show from the Beginning for the benefit of Youth in the Agriculture Industry and Continuing the Tradition of Showing

Youth throughout the country have been shown to benefit through organizations focused on leadership and real-world tangible skills. Organizations such as the National FFA Organization and 4-H Youth Development are key organizations for this growth. Livestock shows are another layer to these organizations and youth development. “Livestock shows provide interactions through different environments that expose participants to different cultures,” (Davis, 1998). For these reasons it is important to provide more opportunities to the next generation of agriculture. There are currently 28 different livestock jackpots within the state of California throughout one year, (CJLA, 2020). Due to the large geographic landscape of California, additional options and sizes of shows are necessary for all who wish to participate in jackpots to have the opportunity. For this reason, the No Limit, High Roller, Livestock Show, will be created

Local Health Department Provision of WIC Services Relative to Local “Need”— Examining 3 States and 5 Years

Great variation exists in the nature of LHD service delivery and it varies, in part, relative to jurisdiction population size. Larger LHD jurisdictions may achieve an economy of scale in WIC service delivery that is not matched in smaller areas. Overall, we found that WIC service provision appears relatively consistent across study states and in the presence of increasing need, with greater responsiveness to need in urban areas. As demand for some preventive services increases LHDs in rural areas may need greater support than LHDs in large jurisdictions for meeting local demand. Unlike WIC, LHD-provided services that have less consistently maintained service-delivery guidelines may have a harder time responding to increasing need. The relative consistency of a federally-funded program such as WIC may serve as a good baseline for further study of less consistently delivered programs among LHDs. LHD service statistics can serve as useful data sources in measuring volume of service delivery relative to need

The Effect of Sleep Quality on Profile of Mood, Physiological Responses, and Athletic Performance

While the extent of an athlete’s physiological adaptations varies due to a range of external factors, sleep deprivation has been proven to negatively affect one’s training and, ultimately, one’s overall athletic success. Sleep deprivation, either for a short or prolonged period of time, also leads to adverse psychological effects, as measured by the alteration of genes in the brain. PURPOSE: This study investigates how both normal sleep and sleep deprivation affect an athlete’s recovery, overall skeletal muscle performance, and mental state. METHODS: Using a randomized-crossover design, 7 healthy individuals, 3 males and 4 females, with at least 1 year of experience in resistance training participated in this study. Exercise testing sessions were identical in nature and took place in the morning following either 3 consecutive nights of 8 hours of sleep (normal) or 3 consecutive nights of 5 hours of sleep (deprivation). Pittsburgh sleep quality index (PSQI) and profile of mood state were collected each morning. Following warm-up, subjects performed the following tests in this order: 1 set of 15 repetitions of a maximal voluntary knee extension/flexion contraction at 300°•s-1, 4 repetitions of 4 seconds of isometric knee extension with EMG data collected, and 1 set of 50 repetitions of a maximal voluntary knee extension/flexion contraction at 60°•s-1. RESULTS: Although some variables in POMS and PSQI were statistically significantly (P\u3c0.05), no statistically significant muscular performance differences were found in normal sleep trials compared with sleep deprivation trials. However, there were trends toward decreased performance during sleep deprivation protocols. In sleep deprivation protocols peak extension and flexion power at 300°•s-1 were decreased 6.8% and 5.5% respectively. Peak torque and average torque during isometric testing decreased 15% and 10% respectively. Similarly, during the 50 repetitions at 60°•s-1, peak torque flexion and extension dropped 4.3% and 4.6% respectively. CONCLUSION: Our hypothesis that sleep deprivation has adverse effects was partially supported in the limited number of subjects, indicating that in an athletic setting, decreased sleep time might be detrimental with various types of athletic performance and one’s psychological well-being

Road verge vegetation and the capture of particulate matter air pollution

Urban air quality is considered a major issue in cities worldwide, with particulate matter (PM) recognised as one of the most harmful pollutants regarding human health. The use of plants to act as air filters and immobilise PM has been identified as a potential method to improve the air quality in these areas. The majority of the work has focused on trees, with the application of shrub and herbaceous species largely overlooked. Two contrasting leaf morphologies from a shrub and herbaceous plant species were sampled at four locations across Southampton (UK), from varying traffic conditions. Samples were analysed for the mass of PM captured, particle size, and elemental composition. These analyses were used to characterise the different sites and the plants’ effectiveness at immobilisation of PM. Captured PM mass was shown to be directly related to traffic density, with greater traffic density leading to higher levels of captured PM. PM origins were attributed to emissions from vehicles and the resuspension of particles by vehicle movement. The bulk of the PM mass was shown to originate from natural, crustal sources including large proportions of Al, Si, and/or Ca. Increases in elements from anthropogenic enhancement (such as Fe and Zn) were related to high traffic density. Particle size analysis identified that, despite the use of standard leaf-washing protocols with a final 2.5 µm filter, PM was dominated by fine particles (<2.5 µm physical diameter), with particles >10 µm rare. Bramble leaves were calculated to have a species-specific deposition velocity 0.51 cm s−1 greater than ivy, with deposition velocities calculated at 1.8 and 1.3 cm s−1 for ivy and 2.3 and 1.8 cm s−1 for bramble at Redbridge Road and Brinton’s Road, respectively. These values can allow for the more accurate modelling and estimation of the PM removal abilities of these plants

Salmon plasma protein to inhibit protease enzymes and enhance surimi gelation

The ability of salmon plasma protein (SPP) obtained from Chinook salmon at the Klaskanine Fish Hatchery (Astoria, OR) to inhibit protease enzymes found in Pacific whiting (PW) surimi and salmon mince as well as the effect of SPP on the gelation properties of PW surimi under various heating conditions was investigated. The first study focused on the biochemical aspects of SPP, including enzyme and autolysis inhibition, stability, and molecular weight determination through inhibitor staining. The second study focused on the effects of SPP on PW surimi gelation using various heating treatments to isolate the activity of both protease enzymes and transglutaminase (TGASE) The results indicated that SPP contains both cysteine and serine protease inhibitors and effectively inhibits autolysis in both PW surimi and salmon mince. As little as 0.25% SPP inhibited PW surimi autolysis by about 90%, suggesting that low concentrations SPP could be effective when added to PW surimi. In addition, both cysteine and serine protease inhibitors in SPP exhibited good thermal, pH and salt stability. As little as 0.5% addition of SPP significantly improved the gel strength of PW surimi that was rapidly heated to 60°C and held at that temperature for 30 min before rapidly heating to 90°C in order to isolate protease enzyme activity. Analysis of TCA-soluble peptide analysis showed no significant proteolytic activity in this gel after SPP was added. One percent addition of SPP was shown to significantly improve the gel strength of PW surimi that was held at 25°C for 2 hr before being rapidly heated to 90°C in order to isolate TGASE activity. This suggests that SPP may contain its own TGASE activity in addition to endogenous TGASE present in PW surimi. These results were confirmed by SDS PAGE and scanning electron microscopy

Education in America: The Views of Millennials

This GenForward report presents the views of young adults between the ages of 18 and 34 on education in the United States. We asked our nationally representative and diverse sample of young adults to provide their evaluations of their own school system, their thoughts about what makes a good school, their preferences regarding proposals for reforming education, their perceptions about issues of equity in schools, and their beliefs about the promise and challenges of higher education in America today. Who better to assess the strengths and weaknesses of our educational systems than those most proximate to the American educational experience? Many Millennials recently graduated from high school, while some are currently pursuing higher educational opportunities and/or navigating educational systems for their children