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Background: SPAK (Ste20p-related proline alanine-rich kinase) phosphorylates and activates NKCC1 (Na-K-2Cl cotransporter) in the presence of another serine/threonine kinase WNK4 (With No lysine (K)). However, whether or not the docking of SPAK to NKCC1 is a requirement for cotransporter activation has not been fully resolved. Methods: We mutated both SPAK binding motifs in the amino-terminal tail of NKCC1 and tested the interaction between SPAK and NKCC1 using a semi in vivo yeast two-hybrid assay, 32P-ATP in vitro phosphorylation assays, and 86Rb+ uptake (a K+ congener) assays in heterologously expressed Xenopus laevis oocytes. We also used site-directed mutagenesis to identify the principle phospho-regulatory threonine residues in the amino-terminal tail of NKCC1. Results: A single SPAK binding motif is necessary for isotonic NKCC1 activation. Mutation of the phenylalanine (F) residue within the motif abrogates binding and function. Phosphorylation of the cotransporter is markedly reduced in the absence of SPAK docking to NKCC1. Truncations of internal regions of the amino-terminus of NKCC1 do not disrupt protein structure enough to affect cotransporter function. Threonine residues (T206 and T211) are both identified as phospho-regulatory sites of NKCC1 function. Conclusion: We demonstrate that physical docking of SPAK to NKCC1 is necessary for cotransporter activity under both baseline and hyperosmotic conditions. We identify T206 and T211 as major phospho-acceptor sites involved in cotransporter function, with T206 common to two separate regulatory pathways: one involving SPAK, the other involving a still unknown kinase that is responsive to forskolin/PKA stimulation. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Previous work from our laboratory and others has established that Ste-20-related proline-alanine-rich kinase (SPAK/PASK) is central to the regulation of NKCC1 function. With no lysine (K) kinase (WNK4) has also been implicated in the regulation of NKCC1 activity through upstream activation of SPAK. Because previous studies from our laboratory also demonstrated a protein-protein interaction between SPAK and apoptosis-associated tyrosine kinase (AATYK), we explore here the possibility that AATYK is another component of the regulation of NKCC1. Heterologous expression of AATYK1 in NKCC1-injected Xenopus laevis oocytes markedly inhibited cotransporter activity under isosmotic conditions. Interestingly, mutation of key residues in the catalytic domain of AATYK1 revealed that the kinase activity does not play a role in the suppression of NKCC1 function. However, mutagenesis of the two SPAK-binding motifs in AATYK1 completely abrogated this effect. As protein phosphatase 1 (PP1) also plays a central role in the dephosphorylation and inactivation of NKCC1, we investigated the possibility that AATYK1 interacts with the phosphatase. We identified a PP1 docking motif in AATYK1 and demonstrated interaction using yeast-2-hybrid analysis. Mutation of a key valine residue (V1175) within this motif prevented protein-protein interaction. Furthermore, the physical interaction between PP1 and AATYK was required for inhibition of NKCC1 activity in Xenopus Iaevis oocytes. Taken together, our data are consistent with AATYK1 indirectly inhibiting the SPAK/WNK4 activation of the cotransporter by scaffolding an inhibitory phosphatase in proximity to a stimulatory kinase. [ABSTRACT FROM AUTHOR]

The article argues that national AIDS commissions have not been successful and provides suggestions for other responses. The article questions the way in which multisector coordination has been implemented, with AIDS prevention and treatment efforts organized around a disparate collection of agencies. AIDS should be treated as a disease, not a social problem. Public health skills must be taught to sex workers and other high risk groups. The author advocates private funding or at least direct control of public funds going to the national commission.

In the present study, we have demonstrated functional interaction between Ste20-related proline-alanine-rich kinase (SPAK), WNK4 [with no lysine (K)], and the widely expressed Na+-K+-2Cl- cotransporter type 1 (NKCC1). NKCC1 function, which we measured in Xenopus Iaevis oocytes under both isosmotic (basal) and hyperosmotic (stimulated) conditions, was unaffected when SPAK and WNK4 were expressed alone. In contrast, expression of both kinases with NKCC1 resulted in a significant increase in cotransporter activity and an insensitivity to external osmolarity or cell volume. NKCC1 activation is dependent on the catalytic activity of SPAK and likely also of WNK4, because mutations in their catalytic domains result in an absence of cotransporter stimulation. The results of our yeast two-hybrid experiments suggest that WNK4 does not interact directly with NKCC1 but does interact with SPAK. Functional experiments demonstrated that the binding of SPAK to WNK4 was also required because a SPAK-interaction-deficient WNK4 mutant (Phe997Ala) did not increase NKCC1 activity. We also have shown that the transport function of K+-Cl- cotransporter type 2 (KCC2), a neuron-specific KCl cotransporter, was diminished by the expression of both kinases under both isosmotic and hyposmotic conditions. Our data are consistent with WNK4 interacting with SPAK, which in turn phosphorylates and activates NKCC1 and phosphorylates and deactivates KCC2. [ABSTRACT FROM AUTHOR]

The article focuses on the thought that rural development holds the potential to stem much of the urban growth by reducing levels of rural-to-urban migration. There is a debate arising about what extent rural development can reduce migration less than half of projected urban growth. Urban growth is not occurring in proportion to existing income groupings. Average income levels in urban squatter settlements can be lower than those in some rural areas. Therefore, income by itself is an unsuitable comparaitve measure of urban and rural standards.

A letter to the editor is presented in response to a Joint Funding Platform being formed by the Global Alliance for Vaccines and Immunisation (GAVI), the Global Fund, and the World Bank.

This paper is on determination of the thermal conductivities of several hydrocarbon base oils by means of non-equilibrium molecular dynamics simulations using two different force fields. It aims to explore a simulation-based method for lubricant molecular design and analysis concerning heat transfer in electrical vehicle lubrication. Argon was analyzed as a reference for method evaluation, and the results reveal that the calculated conductivity strongly depends on the size of the computational domain. However, for hydrocarbon base oils, the dependence on computation domain size is less prominent as the domain size increases. The method of direct calculation in a sufficiently large computation domain and that of reciprocal extrapolation with data calculated in a much smaller domain are both applicable, and each has a certain value in oil conductivity calculation. The calculated conductivities show certain overpredictions when compared with experimentally measured results, and the overprediction factor is related to number of carbon atoms of the liquid molecules. The results reveal that the thermal conductivity of a single-chain hydrocarbon liquid is linearly proportional to the number of carbon atoms. While each additional branch increases thermal conductivity slightly, the presence of multiple branches reduces it from the ideal linear relationship. A set of equations was formulated to correlate hydrocarbon liquid thermal conductivity with molecular characteristics in terms of number of carbon atoms and number of branches. [ABSTRACT FROM AUTHOR]

The article refutes the arguments raised by Nicholas Cunningham concerning the commentary on the mythology of international health planning. The author argues that Cunningham has misrepresented his points. On the issue of rural development, he asserts that development projects invariably operate at levels of resource and skill input. He further contends that the said projects do not constitute development because they do not widely effect distribution. On the issue of prevention, he points out that the cost-effectiveness of measles immunization has been questioned because of its massive expansion without evaluation.

The article refutes the arguments raised by Oscar Gish concerning the commentary on the mythology of international health planning. The author clarifies that he is concerned with planning as a problem-solving or design process. He adds that his commentary attempts to illustrate how myths prohibit the application of the said problem-solving process. On the issue of medical aids, he emphasizes that Gish has overemphasized the state of affairs in manpower design and performance evaluation. On the issue of compulsory service, he asserts that health can only be improved through the willingness of people to evaluate and discard old concepts and myths.

A letter to the editor is presented in response to the article on the role of Joint United Nations Programme on HIV/AIDS (UNAIDS) in global health in the June 18, 2011 issue.

A letter to the editor is presented in response to the article "AIDS: Lessons Learnt and Myths Dispelled," by P. Piot and colleagues in the July 18, 2009 issue.

The author reflects on the creation of UNAIDS, which is a joint United Nations program on HIV and AIDS which was founded because involved individuals determined that HIV is an exceptional disease. He suggests that if there is a United Nations program for HIV perhaps there should be one for other diseases including pneumonia. He argues that far too much is spent on HIV in comparison to other diseases and that this is damaging health systems.

A letter to the editor is presented in response to the article “Malaria control needs mass distribution of insecticidal bednets,” by A. Teklehaimanot, J. D. Sachs and C. Curtis in the June 30, 2007 issue.

The article presents the author's opinions on an International Health Partnership which was launched by Great Britain's Prime Minister Gordon Brown in an effort to accelerate progress towards achieving the United Nations millenium goals for health. Arguments are presented which suggest that programs such as the partnership which give money to countries through disease specific global programs might make good press but do not help the countries, and that what will help countries with inadequate health care is strengthened national healthcare systems that can deliver the range of services that countries need, according to their priorities, not those of international body groups.

Recent work from our laboratory has identified an association between the widely expressed NKCCl and two Ste20p-like serine/threonine kinases, SPAK and OSR1. Activation of the widely expressed NKCCl is mediated by SPAK/OSR1 phosphorylation. Importantly, these kinases are themselves under the control of upstream kinases such as the with-no-lysine kinases, WNK1 and WNK4, suggesting a cascade of phosphorylation events involving multiple kinases. We also recently demonstrated protein-protein interaction between a putative apoptosis-associated tyrosine kinase (AATYK1) and SPAK, and identified two putative (R/KFxV/I) binding motifs within the regulatory domain of AATYK1. Here we show that heterologous expression of AATYK1 and NKCCl in Xenopus laevi s< oocytes exhibits a significant reduction in cotransporter activity. Interestingly, this effect can be reversed by overexpressing SPAK and WNK4. Mutation of key residues in the catalytic domain of AATYK1 reveals that the kinase activity of AATYK1 does not play a role in the suppression of NKCCl function. In contrast, mutagenesis of two SPAK binding motifs in AATYK1 completely abrogated the inhibition of cotransporter activity. Along with a SPAK/AATYK1 interaction, yeast two-hybrid experiments demonstrated a strong interaction between protein phosphatase 1 (PP1) and AATYK1, which could be interrupted by mutation of a key valine residue (V1175) in a putative PP1 binding motif. Functional experiments demonstrated that not only the binding of SPAK, but also PP1 to AATYK1 was required for inhibition of NKCC1 activity. Taken together, our data are consistent with AATYK1 indirectly inhibiting cotransporter activity by scaffolding an inhibitory phosphatase (PP1) in proximity to a stimulatory kinase (SPAK), thus allowing PP1 to dephosphorylate SPAK, resulting in a suppression of SPAK/WNK4 activation of the cotransporter. [ABSTRACT FROM AUTHOR]

The secretory Na-K-2Cl cotransporter, NKCCl, is highly expressed on the basolateral membrane of Cl--secreting epithelia such as salivary, sweat and lacrimal glands as well as lung, stomach, colon, where it participates to salt/fluid secretion. In all cells where the cotransporter is expressed, NKCCl is activated by cell shrinkage and participates in cell volume maintenance and regulation. The isosmotic activation NKCCl by the serine/threonine kinases SPAK (Ste20p-related proline alanine-rich kinase) and WNK4 (with no lysine (K)) requires a functional interaction between the kinases and the cotransporter. The specificity of a kinase to a particular substrate can be significantly increased by a physical docking between the two proteins. We previously identified a single putative SPAK binding motif (R/KFxV/I) in the carboxy terminus of WNK4, and two SPAK binding motifs in the amino terminus of NKCCl. In this study, we demonstrate that a single RFxV motif in the N-terminal tail of NKCCl is necessary for isotonic cotransporter activation, and that an alanine substitution of the conserved phenylalanine (F) residue within the motif abrogates cotransporter activity in NKCCl-injected Xenopus laevis oocytes. We also correlate the in vitro phosphorylation of NKCCl by SPAK with the in vivo isosmotic activation of the cotransporter. Indeed, in the absence of kinase docking to the amino terminus of NKCCl, the rate of kinase phosphorylation of the cotransporter is markedly reduced. Therefore, our data demonstrate that SPAK binding (or docking) to the N-terminus of NKCCl is a pre-requisite for cotransporter stimulation by the kinase. Interestingly, whereas physical binding is necessary for cotransporter activation, the distance between the docking site and the phosphorylation sites appears to be unimportant. [ABSTRACT FROM AUTHOR]

The article presents opinion on whether the amount of money which is being spent on the treatment of HIV worldwide is excessive. Arguments supporting the idea that it is excessive are presented and supported by data from the Organization for Economic Cooperation and Development. The data showed that 21% of all health aid was allocated to HIV in 2004, up from 8% in 2000, and could exceed 25% in 2007, despite the fact that HIV constitutes only 5% of disease in low and middle income countries worldwide.

A letter to the editor is presented in response to the article "Mortality of HIV-1 infected patients in the first year of antiretroviral therapy: comparison between low-income and high-income countries," from the Antiretroviral Therapy in Lower Income Countries Collaboration and ART Cohort Collaboration groups, in the March 11, 2006 issue.

Our recent studies demonstrate that SPAK (Ste20p-related Proline Alanine-rich Kinase), in combination with WNK4 [With No lysine (K) kinase], phosphorylates and stimulates the Na-K-2Cl cotransporter (NKCC1), whereas catalytically inactive SPAK (K104R) fails to activate the cotransporter. The catalytic domain of SPAK contains an activation loop between the well-conserved DFG and APE motifs. We speculated that four threonine residues (T231, T236, T243, and T247) in the activation loop might be sites of phosphorylation and kinase activation; therefore, we mutated each residue into an alanine. In this report, we demonstrate that coexpression of SPAK (T243A) or SPAK (T247A) with WNK4 not only prevented, but robustly inhibited, cotransporter activity in NKCC1-injected Xenopus laevis oocytes. These activation loop mutations produced an effect similar to that of the SPAK (K104R) mutant. In vitro phosphorylation experiments demonstrate that both intramolecular autophosphorylation of SPAK and phosphorylation of NKCC1 are significantly stronger in the presence of Mn2+ rather than Mg2+. We also show that SPAK activity is markedly inhibited by staurosporine and K252a, partially inhibited by N-ethylmaleimide and diamide, and unaffected by arsenite. OSR1, a kinase closely related to SPAK, exhibited similar kinase properties and similar functional activation of NKCC1 when coexpressed with WNK4. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor in response to the article"Achieving the WHO/UNAIDS antiretroviral treatment 3 by 5 goal: what will it cost?" by J. P. Gutierrez et al. in the July 3, 2004 issue.

Activity of heterologously expressed NKCC1 was analyzed under basal and activated conditions in the presence and absence of binding of Ste20-related prolinealanine-rich kinase (SPAK). Mutant NKCC1 that lacks the ability to bind to this kinase showed K[sup +] transport function identical to wild-type NKCC1. Thus, preventing the binding of the kinase to the cotransporter does not affect cotransporter function. In contrast, several experiments suggest a possible role for SPAK as a scaffolding protein. First, Western blot analysis revealed the presence, and in some tissues abundance, of truncated forms of SPAK and OSR1 in which the kinase domains are affected and thus lack kinase activity. Second, a yeast two-hybrid screen of proteins that interact with the regulatory (binding) domain of SPAK identified several proteins all involved in cellular stress pathways. Third, p38, one of the three major MAPKs, can be coimmunoprecipitated with SPAK and with NKCC1 in an activitydependent manner. The amount of p38 coimmunoprecipitated with the kinase and the cotransporter significantly decreases upon cellular stress, whereas the interaction of the kinase with NKCC1 remains unchanged. These findings suggest that cation-chloride cotransporters might act as "sensors" for cellular stress, and SPAK, by interacting with the cotransporter, serves as an intermediate in the response to cellular stress. [ABSTRACT FROM AUTHOR]

Deafness can result from a variety of gene defects. Some genes involved in the physiology of hearing encode membrane transporters that regulate the ionic composition of the fluid bathing the inner ear. The endolymph is an extracellular fluid with an atypical composition that resembles the intracellular milieu, high in K+ and low in Na+. Recent studies have emphasized the prominent role of K+ channels in endolymph secretion and mechanical transduction. Coupled electroneutral transport of Na+, K+ and Cl- is mediated by two isoforms of the Na-K-2Cl co-transporter: the absorptive isoform BSC1 (also called NKCC2, encoded by Slc12a1 in mouse) that is exclusively expressed in kidney; and BSC2/NKCC1 (encoded by Slc12a2 in mouse), the secretory isoform which has a wider pattern of expression including epithelia, muscle cells, neurons and red blood cells. These co-transporters share 57% homology at the amino acid level and are pharmacologically inhibited by loop diuretics. There is functional and histochemical evidence for the presence of the secretory isoform of the Na-K-2Cl co-transporter in gerbil, rat and rabbit inner ear. We disrupted mouse Slc12a2 and report here that Slc12a2-/- mice are deaf and exhibit classic shaker/waltzer behaviour, indicative of inner-ear defects. We localized the co-transporter to key secreting epithelia of the mouse inner ear and show that absence of functional co-transporter leads to structural damages in the inner ear consistent with a decrease in endolymph secretion. [ABSTRACT FROM AUTHOR]

Heavy duty (HD) vehicles are projected to be the largest fuel-use subsector in transportation, with current demand for diesel fuel projected to grow 30% by 2040. Historically, a primary strategy for increasing diesel engine efficiency has been to increase peak cylinder pressure (PCP). However, increasing PCP imparts greater mechanical and thermal loads on engine components and materials. In recent years, the material property limits for many components have been reached and further increases in PCP above ∼20 MPa have been difficult, while still maintaining the necessary affordability and longevity of on-road HD diesel engines. This paper reviews the historical evolution and major metallurgical advancements of high temperature materials in HD on road diesel engines (10–15 L displacement) up to the current state of the art, focusing on materials in the engine block, cylinder heads, pistons, valves, and exhaust components. These components cover a wide range of material classes, including cast iron, ferritic steel, austenitic steel, titanium alloys, nickel based super-alloys, and high temperature coatings. The microstructural degradation and failure mechanisms of the materials associated with the complex mechanical and thermal loading during service are discussed and key areas for future materials research are suggested that overcome technical barriers. [ABSTRACT FROM AUTHOR]

Multilayer, graded ceramic/metal coatings were prepared by an air plasma spray method on Ti-6Al-4V, 4140 steel and graphite substrates. The coatings were designed to provide thermal barriers for diesel engine pistons to operate at higher temperatures with improved thermal efficiency and cleaner emissions. A systematic, progressive variation in the mixture of yttria-stabilized zirconia and bondcoat alloys (NiCoCrAlYHfSi) was designed to provide better thermal expansion match with the substrate and to improve thermal shock resistance and cycle life. Heat transfer through the layers was evaluated by a flash diffusivity technique based on a model of one-dimensional heat flow. The aging effect of the as-sprayed coatings was captured during diffusivity measurements, which included one heating and cooling cycle. The hysteresis of thermal diffusivity due to aging was not observed after 100-h annealing at 800 °C. The measurements of coatings on substrate and freestanding coatings allowed the influence of interface resistance to be evaluated. The microstructure of the multilayer coating was examined using scanning electron microscope and electron probe microanalysis. [ABSTRACT FROM AUTHOR]

Piston skirt coatings are used in heavy-duty diesel engines for reduction of friction, noise, and scuffing. This study explored the friction and wear behavior of two piston skirt coatings, a polymer-graphite composite coating with a manganese phosphate (Mn-P) interlayer and a standalone Mn-P coating, in API CJ-4 and candidate PC-11 (CK-4 and FA-4) diesel engine oils. Two test configurations were used: conformal contact similar to that in an actual engine to examine the friction behavior under normal operating conditions, and point contact to study the wear performance under extreme pressure conditions. Results suggest that CK-4 could replace CJ-4 without wear penalty but FA-4 may post a potential wear challenge. The standalone Mn-P coating showed little friction benefit in the conformal contact, but made a detrimental impact on wear performance of the piston skirt in the point contact. It is hypothesized that the hard Mn-P grains worn off the coating acted as abrasive at the sliding interface to promote third-body abrasion accelerating the material removal. The polymer-graphite composite coating reduced the friction by 15–30% in the conformal contact tests in the three engine oils. However, in the point contact sliding, the polymer-graphite coating caused a higher wear rate, which is attributed to the Mn-P interlayer releasing hard particles after the top coat was worn through. [ABSTRACT FROM AUTHOR]

Peripheral neuropathy associated with agenesis of the corpus callosum (ACCPN) is a severe sensorimotor neuropathy associated with mental retardation, dysmorphic features and complete or partial agenesis of the corpus callosum. ACCPN is transmitted in an autosomal recessive fashion and is found at a high frequency in the province of Quebec, Canada. ACCPN has been previously mapped to chromosome 15q. The gene SLC12A6 (solute carrier family 12, member 6), which encodes the K[SUP+]-CI[SUP-] transporter KCC3 and maps within the ACCPN candidate region, was screened for mutations in individuais with ACCPN. Four distinct protein-truncating mutations were found: two in the French Canadian population and two in non-French Canadian families. The functional consequence of the predominant French Canadian mutation (2436deIG, Thr813fsX813) was examined by heterologous expression of wildtype and mutant KCC3 in Xenopus laevis oocytes; the truncated mutant is appropriately glycosylated and expressed at the cellular membrane, where it is non-functional. Mice generated with a targeted deletion of Sic12a6 have a locomotor deficit, peripheral neuropathy and a sensorimotor gating deficit, similar to the human disease. Our findings identify mutations in SLC12A6 as the genetic lesion underlying ACCPN and suggest a critical role for SLC12A6 in the development and maintenance of the nervous system. [ABSTRACT FROM AUTHOR]

Several letters to the editor are presented in response to articles in previous issues including "Lessons for Health Educators," by Jeannette J. Simmons, "Screening for Hypertension By Optometrists," by John R. Pierce and Robert N. Kleinstein, and "The Case for a National Health Service," by Dr. Milton Terris.