The human ecto-ATPase (NTPDase 2) contains conserved motifs including five apyrase conserved regions (ACRs) and four conserved regions (CRs) as well as conserved lysine and arginine residues that are also present in other cell surface E-NTPDases. Some of the positively charged amino acids may be involved in ATP binding. The protein also contains six potential N-linked glycosylation sites. Results obtained with seven lysine and six arginine mutants indicate the importance of K62 that is located in CR1, K182, which is downstream of ACR3, and R155, which immediately follows CR3. Mutation of asparagine at the six potential N-linked glycosylation sites individually to glutamine established the importance of N64 in CR1 and N443 in ACR5 in protein function and expression. Mutation of N64, which is conserved in all cell surface NTPDases, results in the expression of an unstable protein, the activity of which is only manifested in the presence of concanavalin A. Both K62 and N64 reside in CR1 that is conserved in all cell surface NTPDases. In the sequence of the CR1 of human ecto-ATPase, 58WPADKENDTGIV69, 65DTG67 is similar to the phosphate-binding motif (DXG) in ACR1 and 4. The D65A and G67A mutants have reduced protein expression and activity. Mutations of other residues in CR1 to alanine led to partial to complete loss of protein expression and activity except for P59. The alanine mutants of the three acidic amino acid residues, D61, E63, and D65, all have decreased affinity for divalent ions. D61 can be substituted by glutamate, but E63 appears to be invariable. Taken together, these results indicate that CR1, which follows ACR1 in the cell surface NTPDases, is an essential structural element in these enzymes.

Background: The changing diabetes in children (CDiC) project is a public-private partnership implemented by Novo Nordisk, to improve access to diabetes care for children with type 1 diabetes. This paper outlines the findings from an evaluation of CDiC in Bangladesh and Kenya, assessing whether CDiC has achieved its objectives in each of six core program components.Research design and methods: The Rapid Assessment Protocol for Insulin Access (RAPIA) framework was used to analyze the path of insulin provision and the healthcare infrastructure in place for diagnosis and treatment of diabetes. The RAPIA facilitates a mixed-methods approach to multiple levels of data collection and systems analysis. Information is collected through questionnaires, in-depth interviews and focus group discussions, site visits, and document reviews, engaging a wide range of stakeholders (N = 127). All transcripts were analyzed thematically. Results:The CDiC scheme provides a stable supply of free insulin to children in implementing facilities in Kenya and Bangladesh, and offers a comprehensive package of pediatric diabetes care. However, some elements of the CDiC program were not functioning as originally intended. Transitions away from donor funding and toward government ownership are a particular concern, as patients may incur additional treatment costs, while services offered may be reduced. Additionally, despite subsidized treatment costs, indirect costs remain a substantial barrier to care. Conclusion:Public-private partnerships such as the CDiC program can improve access to life-saving medicines. However, our analysis found several limitations, including concerns over the sustainability of the project in both countries. Any program reliant on external funding and delivered in a high-turnover staffing environment will be vulnerable to sustainability concerns.child, delivery of health care, low-and middle-income countries, public-private sector partnerships, type 1 diabetes

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