Characterisation of the reaction of 1,4-phenylenebismaleimide with Ca²⁺-ATPase and elucidation of the intramolecular crosslink site

The SR Ca²⁺-ATPase is an ATP driven pump that removes calcium from the sarcoplasm and myofibrils to allow muscle relaxation. The sulfhydryl crosslinker, 1,4- phenylenebismaleimide, reacts with Ca²⁺-ATPase (110 kD) to form a species with an apparent molecular weight of 125 kD, as well as dimers and high order oligomers, on SDS-P AGE. During the course of this study we have optimised and characterised the reaction of 1,4-phenylenebismaleimide with SR Ca²⁺-ATPase to produce the 125 kD species that is reminiscent of an E 125 species formed by intramolecular crosslink with glutaraldehyde. The glutaraldehyde crosslink involves the active site Lys 492 and Arg 678, in a zero distance link that overlaps with the ATP binding pocket, since it can be inhibited by nucleotides. It has been previously shown that the putative intramolecular crosslink with 1,4-phenylenebismaleimide is also sensitive to nucleotide binding. We show that the formation of the putative intramolecular crosslink of SR vesicles ( approximately 20 % of ATPase) with 1,4-phenylenebismaleimide is optimum at alkaline pH with micromolar concentrations of the crosslinker. The formation of ATPase dimers and high order oligomers, which were prominent in the reaction with SR vesicles, were eliminated by solubilising in Triton X-100. Under these conditions and in the presence of calcium, two intramolecular crosslinks are formed as seen in the formation of 125 and 130 kD species. The former seems to be in proximity of the y-phosphate and the latter in the β-phosphate region of the ATP binding site according to nucleotide protection studies. In the presence of detergent (Triton X-100) and absence of calcium, only the 125 kD species is formed and requires stabilisation by thapsigargin, a sesquiterpene lactone that binds the transmembrane α-helices. These conditions yield up to 60 % intramolecularly crosslinked ATPase. Trypsin digestion altered the apparent molecular weight of the 125 kD species to 135 kD, suggesting, in accordance with the results of glutaraldehyde crosslink, that the putative intramolecular crosslink 1s between tryptic fragments A and B. [¹⁴C]1,4-phenylenebismaleimide was synthesised to further characterise the reaction and to elucidate crosslinked amino acid residue following protein digestion, radioactive peptide purification, and sequencing. From filtration studies it was evident that a number of sulfhydryl residues were derivatized in both SR vesicles and solubilised Ca²⁺-ATPase. The results suggests that there is very fast reacting set of sulfhydryl groups, which could comprise of sulthydryls from Ca²⁺-ATPase and/or a minor contaminant protein as previous studies have indicated. Only this fast set was reduced by nucleotide binding. In Triton X-100, the total reactive residues increased two-fold and the biphasic nature of the curve showed that the intramolecular crosslink possibly involves a fast reacting sulfhydryl residue and a slow reacting one. Derivatization with [¹⁴C]1,4-phenylenebismaleimide followed by digestion and HPLC analysis revealed radio labelled peaks. Purification and sequencing of the adducts identified 8 reactive cysteines, namely Cys 12, Cys 344, Cys 364, Cys 471, Cys 498, Cys 636, Cys 670 and Cys 674. The cysteines involved in the putative intramolecular crosslink could not be identified but it is proposed that either Cys 471 or Cys 498 crosslink with Cys 670 or Cys 674

Thapsigargin and Dimethyl Sulfoxide Activate Medium P i ↔ HOH Oxygen Exchange Catalyzed by Sarcoplasmic Reticulum Ca 2+ -ATPase

Thapsigargin is a potent inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase. It binds the Ca(2+)-free E2 conformation in the picomolar range, supposedly resulting in a largely catalytically inactive species. We now find that thapsigargin has little effect on medium P(i) HOH oxygen exchange and that this activity is greatly stimulated (up to 30-fold) in the presence of 30% (v/v) Me(2)SO. Assuming a simple two-step mechanism, we have evaluated the effect of thapsigargin and Me(2)SO on the four rate constants governing the reaction of P(i) with Ca(2+)-ATPase. The principal effect of thapsigargin alone is to stimulate EP hydrolysis (k(-2)), whereas that of Me(2)SO is to greatly retard P(i) dissociation (k(-1)), accounting for its well known effect on increasing the apparent affinity for P(i). These effects persist when the agents are used in combination and substantially account for the activated oxygen exchange (v(exchange) = k(-2)[EP]). Kinetic simulations show that the overall rate constant for the formation of EP is very fast (approximately 300 s(-1)) when the exchange is maximal. Thapsigargin greatly stabilizes Ca(2+)-ATPase against denaturation in detergent in the absence of Ca(2+), as revealed by glutaraldehyde cross-linking, suggesting that the membrane helices lock together. It seems that the reactions at the phosphorylation site, associated with the activated exchange reaction, are occurring without much movement of the transport site helices, and we suggest that they may be associated solely with an occluded H+ state

Examining the position of black South African accents in English in Drama Departments within institutions in the Western Cape Province: a case study

This dissertation examines the treatment and perceptions of black South African accents in English in the drama departments of specific institutions of higher learning in the Western Cape Province. The researcher is concerned with the handling of African native cultural identities in the curriculum policy of Drama Departments in higher education institutions in the Western Cape Province. Although the focus of the research is on the institutions that are based in the Western Cape Province, the subjects that are being discussed are representatives of black people in South Africa as a whole. To portray the issue of accents appropriately, the examination does often refer to language as a relatable concept. In the literature of this dissertation, there is ample support for the claim that language differences and disagreements in South Africa are due to the historical and socio-political stance of the European native. After examining the topic of power and social control, the study points out that the native is rarely in control of the interests of the European descendent. Thus, the relationship between the two is immediately assumed before it has even been established. For the purpose of this discussion, ambiguity is appreciated when intimacy between cultures evolves for the greater good. However, it is disregarded when individual voices are diluted into neutral or standard representations of a language. And so the act of reducing black South African accents in English to mirror that of the Euro-American culture robs the natives of their individuality and imprisons them into cultural assimilation. Ultimately, this study intends to identify different groups of black South Africans who speak English with accents. For clarity has divided into two separate groups namely privileged and unprivileged. Accents that are appreciated by white South African English speakers are considered to be privileged, while accents that carry the undertones of any mother-tongue language are recognised in this research study as unprivileged. The study wishes to present this phenomenon as a social issue that will negatively affect the growth of indigenous participation in performance spaces in the Western Cape which the study assumes as the context for the research. The implication of the study is to potentially eliminate issues of inferiority to those black South African accents that have been regarded as less valuable to dramatic performances in the Western Cape, to the extent that platforms of dramatic performances will broaden in order for black South African accents to be prioritised with relevant usag

Prevalence, awareness, control and determinants of hypertension among primary health care professional nurses in Eastern Cape, South Africa

Background: Nurses in primary health care settings are key stakeholders in the diagnosis and management of hypertensive patients. Unfortunately, the working conditions of nurses predispose them to stress, long hours of work, shift duties and unhealthy diets, which are drivers of hypertension. Yet nurses are often overlooked in health screening exercises, primarily because they are assumed to be informed and ‘healthy’. Aim: This study examined the prevalence, awareness, control and determinants of hypertension among professional primary health care nurses in the Eastern Cape Province of South Africa. Setting: This was a cross-sectional survey of 203 professional nurses working at 41 primary health care facilities of the Eastern Cape Province. Methods: A modified WHO STEPwise questionnaire was used for data collection during face-to-face interviews. The information obtained included demographic information, behavioural lifestyles, anthropometric and blood pressure (BP) measurements. Hypertension is defined as an average of two BP ≥ 140/90 mmHg or self-reported history of antihypertensive medication use. Results: The prevalence of hypertension was 52%. Of this, 41% were unaware of their hypertension status. Of those who were aware and on treatment, only 38.1% had a controlled blood pressure. After adjusting for confounders (for physical activity, dietary practices, parity, income and alcohol use), only age and duration of practice were independent predictors of hypertension among the study population. Conclusion: There is a high prevalence of hypertension among the study participants. There is an unexpected low rate of awareness and suboptimal control of blood pressure among the participants. Age is the significant predictor of hypertension among professional nurses in Eastern Cape Province, South Africa. There is an urgent need for the implementation of an effective workplace health programme for nurses in the province

Decentralisation in South Africa : options for District Health Authorities in South Africa

The transfer of authority and responsibility for some public functions from one level of government, especially national government, to a second sphere (provincial) or a third sphere (local governments) has been adopted by many countries with the understanding that such health system decentralisation can help to address political, managerial and operational issues in terms of systemic efficiency and cost-effectiveness. While South Africa’s health system is already structured with a certain degree of decentralisation, the implementation of National Health Insurance (National Health Insurance) and the proposed creation of District Health Authorities as outlined in the Green Paper on NHI, implies that the country will now have to undertake several further steps in decentralising its health system. If poorly designed or ineffectively implemented , decentralisation can exacerbate existing inequalities and inefficiencies and create new challenges and problems. This chapter explores several possibilities for the implementation of a coherent decentralisation system which addresses the health needs of the population. The authors also provide an extensive overview of the various forms and decentralisation and then offer some lessons, caveats and important issues that must be taken into account in the country’s journey to further decentralisation. The potential role of the National Department of Health in a new decentralised system is considered, and several criteria to guide and stagger the decentralisation process are offered. The authors conclude that while decentralisation is not without its disadvantages, decentralisation of health care services in South Africa can make a phenomenal impact on the quality and access to much-needed health services for the most vulnerable populations, particularly women and children and further note that ongoing monitoring and evaluation against set targets will be needed in order to achieve successful implementation of the envisaged NHI-funded health system.http://reference.sabinet.co.za/sa_epublication/healthram2016School of Health Systems and Public Health (SHSPH

The food and nutrition environment at secondary schools in the Eastern Cape, South Africa as reported by learners

CITATION: Okeyo, A. P. et al. 2020. The food and nutrition environment at secondary schools in the Eastern Cape, South Africa as reported by learners. International Journal of Environmental Research and Public Health, 17(11). doi:10.3390/ijerph17114038The original publication is available at https://www.mdpi.com/journal/ijerphOverweight and obesity are growing concerns in adolescents, particularly in females in South Africa. The aim of this study was to evaluate the food and nutrition environment in terms of government policy programs, nutrition education provided, and foods sold at secondary schools in the Eastern Cape province. Sixteen schools and grade 8–12 learners (N = 1360) were randomly selected from three health districts comprising poor disadvantaged communities. Based on age and sex specific body mass index (BMI) cut-off values, 13.3% of males and 5.5% of females were underweight, while 9.9% of males and 36.1% of females were overweight or obese. The main food items purchased at school were unhealthy energy-dense items such as fried flour dough balls, chocolates, candies, and crisps/chips. Nutrition knowledge scores based on the South African food-based dietary guidelines (FBDGs) were poor for 52% to 23.4% learners in Grades 8 to 12, respectively. Female learners generally had significantly higher nutrition knowledge scores compared to their male counterparts (p = 0.016). Questions poorly answered by more than 60% of learners, included the number of fruit and vegetable portions required daily, food to eat when overweight, foods containing fiber, and importance of legumes. It was noted that the majority of teachers who taught nutrition had no formal nutrition training and their responses to knowledge questions were poor indicating that they were not familiar with the FBDGs, which are part of the curriculum. Nutrition assessment as part of the Integrated School Health Program was done on few learners. Overall however, despite some challenges the government national school meal program provided meals daily to 96% of learners. In general, the school food and nutrition environment was not conducive for promoting healthy eating.https://www.mdpi.com/1660-4601/17/11/4038Publishers versio

Piloting a global mentorship initiative to support African emergency nurses

This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited.Mentorship is a relationship whereby a person experienced in the specialty supports and guides a less experienced person following a process of relationship building, engagement and development, self-inquiry and reflection (Seekoe, 2014). It involves regular contact with a critical friend who has a mature level of skills and expertise and can advise and support the mentee’s development in order to empower them and build capacity (Seekoe, 2014). In healthcare this relationship usually has formal recognition. Indeed clinical mentorship of nurse initiated antiretroviral therapy in resource limited settings in South Africa suggests that mentorship increases clinical confidence and improves the quality of nursing care (Green, de Azevedo, Patten, Davies, Ibeto, et al., 2014). Bennet, Paina, Ssengooba, Waswa and Imunya (2013) demonstrated in Kenya and Uganda that mentorship programmes have a positive impact on career development and whether to remain in health research. However, in a global healthcare context, one size does not fit all. Limited examples exist of Afrocentric mentorship arrangements which enhance the personal development and expertise of novice health practitioners however no current mentorship infrastructure exists to support the development of emergency nurses in Africa (Brysiewicz, 2012). This paper explains the creation and pilot of a global mentorship initiative through collaboration to support emergency nurses in Africa and, the potential benefits and limitations of such a quest.Peer reviewe

EXPERIENCES OF FINAL-YEAR NURSING STUDENTS AT A PUBLIC INSTITUTION IN THE EASTERN CAPE PROVINCE REGARDING THEIR PREPAREDNESS TO BECOME PROFESSIONAL NURSES

The purpose of this article is to describe experiences of final-year students at a public college of nursing regarding their preparedness to become registered nurses and to recommend actions to enhance the preparation of nursing students for transition. A qualitative, exploratory and descriptive research design was utilised to reach the objectives of the study of 27 purposively selected final-year nursing students. The results indicated that most of final-year nursing students experienced lack of preparedness to assume the professional nurse’s role with regard to certain factors, including: curriculum-related aspects; and equipment and library resources during nursing students’ training. Some experienced adequate preparedness in relation to role competency such as patient care. Nursing students lacked confidence and expressed apprehension about meeting the performance expectations of the workplace. The researcher recommends the identification of learning needs, their consideration, and contribution to the periodical review of the curriculum; reinstatement of clinical preceptors; standardisation of procedures; formulation of procedure manuals and delegation of finalists for managerial duties

A model for mentoring newly-appointed nurse educators in nursing education institutions in South Africa

Background: South Africa transformed higher education through the enactment of the Higher Education Act (No. 101 of 1997). The researcher identified the need to develop a model for the mentoring of newly-appointed nurse educators in nursing education institutions in South Africa. Objectives: To develop and describe the model for mentoring newly-appointed nurse educators in nursing education institutions in South Africa. Method: A qualitative and theory-generating design was used (following empirical findings regarding needs analysis) in order to develop the model. The conceptualisation of the framework focused on the context, content, process and the theoretical domains that influenced the model. Ideas from different theories were borrowed from and integrated with the literature and deductive and inductive strategies were applied. Results: The structure of the model is multidimensional and complex in nature (macro, mesoand micro) based on the philosophy of reflective practice, competency-based practice andcritical learning theories. The assumptions are in relation to stakeholders, context, mentoring, outcome, process and dynamic. The stakeholders are the mentor and mentee within an interactive participatory relationship. The mentoring takes place within the process with a sequence of activities such as relationship building, development, engagement, reflective process and assessment. Capacity building and empowerment are outcomes of mentoring driven by motivation. Conclusion: The implication for nurse managers is that the model can be used to develop mentoring programmes for newly-appointed nurse educators. </span