SIOP pediatric oncology services Global Mapping Program: Latin American data collection.

The International Society of Paediatric Oncology (SIOP) launched a program to map all pediatric cancer facilities around the world. After the results in Africa were completed, the strategy for data collection for Latin America was revised to improve the accuracy and avoid duplications. In partnership with SIOP, the Sociedad Latino Americana de Oncología Pediátrica (SLAOP) approached their delegates who provided the contacts for a 10-question survey about their institutional capacities. Data were collected by email, online meetings, or telephone calls, and stored in a secure platform. All but one country participated and a high number of centers were recorded.

Access to Methotrexate Monitoring in Latin America: A Multicountry Survey of Supportive Care Capacity.

High-dose methotrexate (HDMTX) is used to treat a broad spectrum of cancers. Methotrexate (MTX) monitoring and adequate supportive care are critical for safe drug administration; however, MTX level timing is not always possible in low- and middle-income countries. The aim of this study was to evaluate HDMTX supportive care capacity and MTX monitoring practices in Latin America (LATAM) to identify gaps and opportunities for improvement. A multicenter survey was conducted among LATAM pediatric oncologists. Twenty healthcare providers from 20 institutions answered the online questionnaire. HDMTX was used to treat acute lymphoblastic leukemia (ALL; 100%), non-Hodgkin lymphoma (84.2%), diffuse large B-cell lymphoma (47.4%), osteosarcoma (78.9%), and medulloblastoma (31.6%). Delays in starting HDMTX infusion were related to bed shortages (47.4%) and MTX shortages (21.1%). MTX monitoring was performed at an in-hospital laboratory in 52%, at an external/nearby laboratory in 31.6%, and was not available in 10.5%. Median interval between sampling and obtaining MTX levels was ≤ 2 h in 45% and ≥ 6 h in 30%, related to laboratory location. Sites without access to MTX monitoring reduced the MTX dose for patients with high-risk ALL or did not include MTX in the treatment of patients with osteosarcoma. Respondents reported that implementation of point-of-care testing of MTX levels is feasible. In LATAM, highly variable supportive care capacity may affect the safe administration of MTX doses. Improving accessibility of MTX monitoring and the speed of obtaining results should be prioritized to allow delivery of full doses of MTX required by the current protocols.

The Triad Na+ Activated Na+ Channel (Nax)-Salt Inducible KINASE (SIK) and (Na+ + K+)-ATPase: Targeting the Villains to Treat Salt Resistant and Sensitive Hypertension.

The Na+-activated Na+ channel (Nax) and salt-inducible kinase (SIK) are stimulated by increases in local Na+ concentration, affecting (Na+ + K+)-ATPase activity. To test the hypothesis that the triad Nax/SIK/(Na+ + K+)-ATPase contributes to kidney injury and salt-sensitive hypertension (HTN), uninephrectomized male Wistar rats (200 g; n = 20) were randomly divided into 4 groups based on a salt diet (normal salt diet; NSD-0.5% NaCl-or high-salt diet; HSD-4% NaCl) and subcutaneous administration of saline (0.9% NaCl) or deoxycorticosterone acetate (DOCA, 8 mg/kg), as follows: Control (CTRL), CTRL-Salt, DOCA, and DOCA-Salt, respectively. After 28 days, the following were measured: kidney function, blood pressure, (Na+ + K+)-ATPase and SIK1 kidney activities, and Nax and SIK1 renal expression levels. SIK isoforms in kidneys of CTRL rats were present in the glomerulus and tubular epithelia; they were not altered by HSD and/or HTN. CTRL-Salt rats remained normotensive but presented slight kidney function decay. HSD rats displayed augmentation of the Nax/SIK/(Na+ + K+)-ATPase pathway. HTN, kidney injury, and kidney function decay were present in all DOCA rats; these were aggravated by HSD. DOCA rats presented unaltered (Na+ + K+)-ATPase activity, diminished total SIK activity, and augmented SIK1 and Nax content in the kidney cortex. DOCA-Salt rats expressed SIK1 activity and downregulation in (Na+ + K+)-ATPase activity in the kidney cortex despite augmented Nax content. The data of this study indicate that the (Na+ + K+)-ATPase activity response to SIK is attenuated in rats under HSD, independent of HTN, as a mechanism contributing to kidney injury and salt-sensitive HTN.

A Systematic Review of High-Dose Methotrexate for Adults with Primary Central Nervous System Lymphoma.

Primary central nervous system lymphoma (PCNSL) is a highly aggressive non-Hodgkin lymphoma that is confined within the CNS. Due to its ability to cross the blood-brain barrier, high-dose methotrexate (HDMTX) is the backbone for induction chemotherapy. This systematic review was conducted to observe outcomes among different HDMTX doses (low, <3 g/m2; intermediate, 3-4.9 g/m2; high, ≥5 g/m2) and regimens used in the treatment of PCNSL. A PubMed search resulted in 26 articles reporting clinical trials using HDMTX for PCNSL, from which 35 treatment cohorts were identified for analysis. The median dose of HDMTX used for induction was 3.5 g/m2 (interquartile range IQR, 3-3.5); the intermediate dose was most frequently used in the studies examined (24 cohorts, 69%). Five cohorts used HDMTX monotherapy, 19 cohorts used HDMTX + polychemotherapy, and 11 cohorts used HDMTX + rituximab ± polychemotherapy. Pooled overall response rate (ORR) estimates for low, intermediate, and high dose HDMTX cohorts were 71%, 76%, and 76%, respectively. Pooled 2-year progression-free survival (PFS) estimates for low, intermediate, and high HDMTX dose cohorts were 50%, 51%, and 55%, respectively. Regimens that included rituximab showed a tendency to have higher ORR and 2-year PFS than those that did not include rituximab. These findings indicate that current protocols utilizing 3-4 g/m2 of HDMTX in combination with rituximab provide therapeutic efficacy in PCNSL.

Insights from a pharmacometric analysis of HDMTX in adults with cancer: Clinically relevant covariates for application in precision dosing.

AIMS: High-dose methotrexate (HDMTX) is an essential part of the treatment of several adult and paediatric malignancies. Despite meticulous supportive care during HDMTX administration, severe toxicities, including acute kidney injury (AKI), may occur contributing to patient morbidity. Population pharmacokinetics provide a powerful tool to predict time to clear HDMTX and adjust subsequent doses. We sought to develop and validate pharmacokinetic models for HDMTX in adults with diverse malignancies and to relate systemic exposure with the occurrence of severe toxicity. METHODS: Anonymized, de-identified data were provided from 101 US oncology practices that participate in the Guardian Research Network, a non-profit clinical research consortium. Modelled variables included clinical, laboratory, demographic and pharmacological data. Population pharmacokinetic analysis was performed by means of nonlinear mixed effects modelling using MonolixSuite. RESULTS: A total of 693 HDMTX courses from 243 adults were analysed, of which 62 courses (8.8%) were associated with stage 2/3 acute kidney injury (43 stage 2, 19 stage 3). A three-compartment model adequately fitted the data. Time-dependent serum creatinine, baseline serum albumin and allometrically scaled bodyweight were clinically significant covariates related to methotrexate clearance. External evaluation confirmed a satisfactory predictive performance of the model in adults receiving HDMTX. Dose-normalized methotrexate concentration at 24 and 48 hours correlated with AKI incidence. CONCLUSION: We developed a population pharmacometric model that considers weight, albumin and time-dependent creatinine that can be used to guide supportive care in adult patients with delayed HDMTX elimination.

Access to care for childhood cancers in India: perspectives of health care providers and the implications for universal health coverage.

BACKGROUND: There are multiple barriers impeding access to childhood cancer care in the Indian health system. Understanding what the barriers are, how various stakeholders perceive these barriers and what influences their perceptions are essential in improving access to care, thereby contributing towards achieving Universal Health Coverage (UHC). This study aims to explore the challenges for accessing childhood cancer care through health care provider perspectives in India. METHODS: This study was conducted in 7 tertiary cancer hospitals (3 public, 3 private and 1 charitable trust hospital) across Delhi and Hyderabad. We recruited 27 healthcare providers involved in childhood cancer care. Semi-structured interviews were audio recorded after obtaining informed consent. A thematic and inductive approach to content analysis was conducted and organised using NVivo 11 software. RESULTS: Participants described a constellation of interconnected barriers to accessing care such as insufficient infrastructure and supportive care, patient knowledge and awareness, sociocultural beliefs, and weak referral pathways. However, these barriers were reflected upon differently based on participant perception through three key influences: 1) the type of hospital setting: public hospitals constituted more barriers such as patient navigation issues and inadequate health workforce, whereas charitable trust and private hospitals were better equipped to provide services. 2) the participant's cadre: the nature of the participant's role meant a different degree of exposure to the challenges families faced, where for example, social workers provided more in-depth accounts of barriers from their day-to-day interactions with families, compared to oncologists. 3) individual perceptions within cadres: regardless of the hospital setting or cadre, participants expressed individual varied opinions of barriers such as acceptance of delay and recognition of stakeholder accountabilities, where governance was a major issue. These influences alluded to not only tangible and structural barriers but also intangible barriers which are part of service provision and stakeholder relationships. CONCLUSION: Although participants acknowledged that accessing childhood cancer care in India is limited by several barriers, perceptions of these barriers varied. Our findings illustrate that health care provider perceptions are shaped by their experiences, interests and standpoints, which are useful towards informing policy for childhood cancers within UHC.

Evaluation of a Pharmacist Led Oral Chemotherapy Clinic: A Pilot Program in the Gastrointestinal Oncology Clinic at an Academic Medical Center.

Oral chemotherapy represents a major patient-centric advancement in therapy convenience. However, ownership of safe and correct administration of these agents requires significant patient education. To address this challenge, an in-person pharmacist-led oral chemotherapy education clinic in gastrointestinal oncology patients within an academic medical center was created and assessed. In this pilot program, a medication-specific quiz was administered to patients before and after education performed by a pharmacist to assess patient understanding of their new oral chemotherapy. A five-question satisfaction survey was also administered at the conclusion of the pharmacist clinic visit. Primary outcome was the percentage difference between pre-and post-education quiz scores. Secondary outcomes included patient satisfaction, time to treatment initiation, and number of pharmacist interventions. Frequencies and medians were used to describe categorical and continuous variables, respectively. Of the 18 patients analyzed, 50% were male and median age was 59.5 years. Approximately 28% had colon cancer, and 61% were treated with capecitabine. The median post-education scores improved from a pre-education score of 75% to 100%. Overall, seventeen of the eighteen patients responded with "strongly agree" to all satisfaction survey statements. An in-person oncology pharmacist-led oral chemotherapy education session demonstrated an improvement in patients' understanding of their new oral chemotherapy treatment.

A health care labyrinth: perspectives of caregivers on the journey to accessing timely cancer diagnosis and treatment for children in India.

BACKGROUND: Cure rates for children with cancer in India lag behind that of high-income countries. Various disease, treatment and socio-economic related factors contribute to this gap including barriers in timely access of diagnostic and therapeutic care. This study investigated barriers to accessing care from symptom onset to beginning of treatment, from perspectives of caregivers of children with cancer in India. METHODS: Semi-structured in-depth interviews were conducted with caregivers of children (< 18 years) diagnosed with cancer in seven tertiary care hospitals across New Delhi and Hyderabad. Purposive sampling to saturation was used to ensure adequate representation of the child's gender, age, cancer type, geographical location and socioeconomic status. Interviews were audio recorded after obtaining informed consent. Thematic content analysis was conducted and organised using NVivo 11. RESULTS: Thirty-nine caregivers were interviewed, where three key themes emerged from the narratives: time intervals to definitive diagnosis and treatment, the importance of social supportive care and the overall accumulative impacts of the journey. There were two phases encapsulating the experiences of the family: referral pathways taken to reach the hospital and after reaching the hospital. Most caregivers, especially those from distant geographical areas had variable and inconsistent referral pathways partly due to poor availability of specialist doctors and diagnostic facilities outside major cities, influence from family or friends, and long travel times. Upon reaching the hospital, families mostly from public hospitals faced challenges navigating the hospital facilities, finding accommodation, and comprehending the diagnosis and treatment pathway. Throughout both phases, financial constraint was a recurring issue amongst low-income families. The caregiver's knowledge and awareness of the disease and health system, religious and social factors were also common barriers. CONCLUSION: This qualitative study highlights and explores some of the barriers to childhood cancer care in India. Our findings show that referral pathways are intrinsically linked to the treatment experience and there should be better recognition of the financial and emotional challenges faced by the family that occur prior to definitive diagnosis and treatment. This information would help inform various stakeholders and contribute to improved interventions addressing these barriers.

Inherited and De Novo Genetic Risk for Autism Impacts Shared Networks.

We performed a comprehensive assessment of rare inherited variation in autism spectrum disorder (ASD) by analyzing whole-genome sequences of 2,308 individuals from families with multiple affected children. We implicate 69 genes in ASD risk, including 24 passing genome-wide Bonferroni correction and 16 new ASD risk genes, most supported by rare inherited variants, a substantial extension of previous findings. Biological pathways enriched for genes harboring inherited variants represent cytoskeletal organization and ion transport, which are distinct from pathways implicated in previous studies. Nevertheless, the de novo and inherited genes contribute to a common protein-protein interaction network. We also identified structural variants (SVs) affecting non-coding regions, implicating recurrent deletions in the promoters of DLG2 and NR3C2. Loss of nr3c2 function in zebrafish disrupts sleep and social function, overlapping with human ASD-related phenotypes. These data support the utility of studying multiplex families in ASD and are available through the Hartwell Autism Research and Technology portal.

Antidiabetic, cytotoxic and antioxidant activities of oil extracted from Acrocomia aculeata pulp.

This study evaluated the hypoglycemic effect of the oil extracted from the Acrocomia aculeata pulp (OPAC) in normoglycemic rats and streptozotocin (STZ), fructose-induced diabetic rat models and its in vitro antioxidant and cytotoxic potential. OPAC (3, 30 or 300 mg/kg, v.o.) significantly decreased (p < 0.05) the high glucose levels induced by a high fructose-diet in rats. Persistent treatment with OPAC for 24 days also reduced the high plasmatic glucose induced by STZ. In normoglycemic animals, OPAC significantly decreased glucose levels. While A. aculeata oil exhibited good in vitro antioxidant activity, no sign of cytotoxicity was observed in LLC-PK1 cells (5-500 µg/mL). OPAC has antidiabetic and antioxidant activities without causing in vitro cytotoxicity.

Inappropriate activity of local renin-angiotensin-aldosterone system during high salt intake: impact on the cardio-renal axis / Atividade inadequada do sistema renina-angiotensina-aldosterona local durante período de alta ingestão de sal: impacto sobre o eixo cardiorrenal

ABSTRACT Although there is a general agreement on the recommendation for reduced salt intake as a public health issue, the mechanism by which high salt intake triggers pathological effects on the cardio-renal axis is not completely understood. Emerging evidence indicates that the renin-angiotensin-aldosterone system (RAAS) is the main target of high Na+ intake. An inappropriate activation of tissue RAAS may lead to hypertension and organ damage. We reviewed the impact of high salt intake on the RAAS on the cardio-renal axis highlighting the molecular pathways that leads to injury effects. We also provide an assessment of recent observational studies related to the consequences of non-osmotically active Na+ accumulation, breaking the paradigm that high salt intake necessarily increases plasma Na+ concentration promoting water retention

RESUMO Apesar de haver uma concordância geral sobre a necessidade de redução na ingestão de sal como questão de saúde publica, o mecanismo pelo qual a alta ingesta de sal deflagra efeitos patológicos sobre o eixo cardiorrenal não está ainda completamente elucidado. Cada vez mais evidencias indicam que o sistema renina-angiotensina-aldosterona (SRAA) seja o principal alvo da alta ingesta de Na+. Uma ativação inadequada do SRAA tecidual pode causar hipertensão e dano ao órgão. Nós revisamos o impacto da dieta com alto teor de sódio sobre o eixo cardiorrenal, destacando as vias moleculares que causam a lesão. Também fizemos uma avaliação de recentes estudos observacionais relacionados às consequências do acúmulo de Na+ não osmoticamente ativo, quebrando assim o paradigma de que a alta ingestão de sódio necessariamente aumenta a concentração sérica de Na+, assim promovendo a retenção de água.

Dissecting copper homeostasis in diabetes mellitus.

Diabetes Mellitus (DM) is characterized by elevated blood glucose levels (hyperglycemia). It can occur due to impaired secretion or action of the hormone insulin, which is produced by pancreatic beta-cells to promote the entry of glucose into the cells. It is known that hyperglycemia has an important role in the production of reactive oxygen species in all types of DM and that an imbalance of transition metal as Cu and Fe plays a pivotal role in stimulating the oxidative stress. Different levels of some transition metals, as Cu, Fe, Mn, and Zn has been reported comparing diabetic animal models with the control group. An increased Cu status is also described in diabetic patients. Homeostasis of Cu depends on distinct proteins, where Cu(I)-ATPases are important transmembrane proteins for acquisition, active transport, distribution and elimination of Cu ions. In this review we first provide an overview of the literature about the relationship between diabetes and copper, the modulation of Cu(I)-ATPases activity and protein expression in DM, to next discuss the alternative treatments for diabetes using Cu chelation. © 2016 IUBMB Life, 69(4):255-262, 2017.

Rare Inherited and De Novo CNVs Reveal Complex Contributions to ASD Risk in Multiplex Families.

Rare mutations, including copy-number variants (CNVs), contribute significantly to autism spectrum disorder (ASD) risk. Although their importance has been established in families with only one affected child (simplex families), the contribution of both de novo and inherited CNVs to ASD in families with multiple affected individuals (multiplex families) is less well understood. We analyzed 1,532 families from the Autism Genetic Resource Exchange (AGRE) to assess the impact of de novo and rare CNVs on ASD risk in multiplex families. We observed a higher burden of large, rare CNVs, including inherited events, in individuals with ASD than in their unaffected siblings (odds ratio [OR] = 1.7), but the rate of de novo events was significantly lower than in simplex families. In previously characterized ASD risk loci, we identified 49 CNVs, comprising 24 inherited events, 19 de novo events, and 6 events of unknown inheritance, a significant enrichment in affected versus control individuals (OR = 3.3). In 21 of the 30 families (71%) in whom at least one affected sibling harbored an established ASD major risk CNV, including five families harboring inherited CNVs, the CNV was not shared by all affected siblings, indicating that other risk factors are contributing. We also identified a rare risk locus for ASD and language delay at chromosomal region 2q24 (implicating NR4A2) and another lower-penetrance locus involving inherited deletions and duplications of WWOX. The genetic architecture in multiplex families differs from that in simplex families and is complex, warranting more complete genetic characterization of larger multiplex ASD cohorts.

Single exposure to cocaine impairs aspartate uptake in the pre-frontal cortex via dopamine D1-receptor dependent mechanisms.

Dopamine and glutamate play critical roles in the reinforcing effects of cocaine. We demonstrated that a single intraperitoneal administration of cocaine induces a significant decrease in [(3)H]-d-aspartate uptake in the pre-frontal cortex (PFC). This decrease is associated with elevated dopamine levels, and requires dopamine D1-receptor signaling (D1R) and adenylyl cyclase activation. The effect was observed within 10min of cocaine administration and lasted for up to 30min. This rapid response is related to D1R-mediated cAMP-mediated activation of PKA and phosphorylation of the excitatory amino acid transporters EAAT1, EAAT2 and EAAT3. We also demonstrated that cocaine exposure increases extracellular d-aspartate, l-glutamate and d-serine in the PFC. Our data suggest that cocaine activates dopamine D1 receptor signaling and PKA pathway to regulate EAATs function and extracellular EAA level in the PFC.

Large-Scale Phenomics Identifies Primary and Fine-Tuning Roles for CRKs in Responses Related to Oxidative Stress.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.

ATP7B activity is stimulated by PKCɛ in porcine liver.

Copper is necessary for all organisms since it acts as a cofactor in different enzymes, although toxic at high concentrations. ATP7B is one of two copper-transporting ATPases in humans, its vital role being manifested in Wilson disease due to a mutation in the gene that encodes this pump. Our objective has been to determine whether pathways involving protein kinase C (PKC) modulate ATP7B activity. Different isoforms of PKC (α, ɛ, ζ) were found in Golgi-enriched membrane fractions obtained from porcine liver. Cu(I)-ATPase activity was assessed in the presence of different activators and inhibitors of PKC signaling pathways. PMA (10(-8) M), a PKC activator, increased Cu(I)-ATPase activity by 60%, whereas calphostin C and U73122 (PKC and PLC inhibitors, respectively) decreased the activity by 40%. Addition of phosphatase λ decreased activity by 60%, irrespective of pre-incubation with PMA. No changes were detected with 2 µM Ca(2+), whereas PMA plus EGTA increased activity. This enhanced activity elicited by PMA decreased with a specific inhibitor of PKCɛ to levels comparable with those found after phosphatase λ treatment, showing that the ɛ isoform is essential for activation of the enzyme. This regulatory phosphorylation enhanced Vmax without modifying affinities for ATP and copper. It can be concluded that signaling pathways leading to DAG formation and PKCɛ activation stimulate the active transport of copper by ATP7B, thus evidencing a central role for this specific kinase-mediated mechanism in hepatic copper handling.

Incidence of acute postoperative cystoid macular edema in clinical practice.

PURPOSE: To measure the incidence of acute postoperative cystoid macular edema (CME) in clinical practice. SETTING: Private clinical practice, Eugene, Oregon, USA. DESIGN: Retrospective chart review. METHODS: The electronic medical records database was searched for the charts of all patients having cataract surgery during a recent 5-year period. Each chart was then examined for a diagnosis of CME within the 3-month postoperative period. RESULTS: From March 1, 2007, through March 31, 2012, 2862 phacoemulsification cataract extraction and intraocular lens implantation procedures were performed. Of these, 3 cases (0.1%) of acute postoperative CME were confirmed by diagnosis within 90 days after surgery. CONCLUSIONS: This chart review found a 0.1% cumulative incidence of acute postoperative CME. Prophylaxis with topical steroidal and nonsteroidal agents may help reduce the incidence of CME. If the incidence of CME in this study applies generally, it would be harder to successfully and ethically perform a randomized trial of medical prophylaxis. FINANCIAL DISCLOSURE: Dr. Packer is a consultant to Advanced Medical Optics, Inc., Allergan, Inc., Bausch & Lomb Surgical, Inc., and Ista Pharmaceuticals, Inc. No other author has a financial or proprietary interest in any material or method mentioned.

Exposure of luminal membranes of LLC-PK1 cells to ANG II induces dimerization of AT1/AT2 receptors to activate SERCA and to promote Ca2+ mobilization.

ANG II is secreted into the lumens of proximal tubules where it is also synthesized, thus increasing the local concentration of the peptide to levels of potential physiological relevance. In the present work, we studied the effect of ANG II via the luminal membranes of LLC-PK(1) cells on Ca(2+)-ATPase of the sarco(endo)plasmic reticulum (SERCA) and plasma membrane (PMCA). ANG II (at concentrations found in the lumen) stimulated rapid (30 s) and persistent (30 min) SERCA activity by more than 100% and increased Ca(2+) mobilization. Pretreatment with ANG II for 30 min enhanced the ANG II-induced Ca(2+) spark, demonstrating a positively self-sustained stimulus of Ca(2+) mobilization by ANG II. ANG II in the medium facing the luminal side of the cells decreased with time with no formation of metabolites, indicating peptide internalization. ANG II increased heterodimerization of AT(1) and AT(2) receptors by 140%, and either losartan or PD123319 completely blocked the stimulation of SERCA by ANG II. Using the PLC inhibitor U73122, PMA, and calphostin C, it was possible to demonstrate the involvement of a PLC→DAG(PMA)→PKC pathway in the stimulation of SERCA by ANG II with no effect on PMCA. We conclude that ANG II triggers SERCA activation via the luminal membrane, increasing the Ca(2+) stock in the reticulum to ensure a more efficient subsequent mobilization of Ca(2+). This first report on the regulation of SERCA activity by ANG II shows a new mechanism for Ca(2+) homeostasis in renal cells and also for regulation of Ca(2+)-modulated fluid reabsorption in proximal tubules.

Mesenchymal stem cell migration is regulated by fibronectin through α5ß1-integrin-mediated activation of PDGFR-ß and potentiation of growth factor signals.

Cell migration during vascular remodelling is regulated by crosstalk between growth factor receptors and integrin receptors, which together coordinate cytoskeletal and motogenic changes. Here, we report extracellular matrix (ECM)-directed crosstalk between platelet-derived growth factor receptor (PDGFR)-ß and α5ß1-integrin, which controls the migration of mesenchymal stem (stromal) cells (MSCs). Cell adhesion to fibronectin induced α5ß1-integrin-dependent phosphorylation of PDGFR-ß in the absence of growth factor stimulation. Phosphorylated PDGFR-ß co-immunoprecipitated with α5-integrin and colocalised with α5ß1-integrin in the transient tidemarks of focal adhesions. Adhesion to fibronectin also strongly potentiated PDGF-BB-induced PDGFR-ß phosphorylation and focal adhesion kinase (FAK) activity, in an α5ß1-integrin-dependent manner. PDGFR-ß-induced phosphoinositide 3-kinase (PI3K) and Akt activity, actin reorganisation and cell migration were all regulated by fibronectin and α5ß1-integrin. This synergistic relationship between α5ß1-integrin and PDGFR-ß is a fundamental determinant of cell migration. Thus, fibronectin-rich matrices can prime PDGFR-ß to recruit mesenchymal cells at sites of vascular remodelling.

Two serine residues control sequential steps during catalysis of the yeast copper ATPase through different mechanisms that involve kinase-mediated phosphorylations.

Ccc2, the yeast copper-transporting ATPase, pumps copper from the cytosol to the Golgi lumen. During its catalytic cycle, Ccc2 undergoes auto-phosphorylation on Asp(627) and uses the energy gained to transport copper across the cell membrane. We previously demonstrated that cAMP-dependent protein kinase (PKA) controls the energy interconversion (Cu)E∼P → E-P + Cu when Ser(258) is phosphorylated. We now demonstrate that Ser(258) is essential in vivo for copper homeostasis in extremely low copper and iron concentrations. The S258A mutation abrogates all PKA-mediated phosphorylations of Ccc2, whereas the S971A mutation leads to a 100% increase in its global regulatory phosphorylation. With S258A, the first-order rate constant of catalytic phosphorylation by ATP decreases from 0.057 to 0.030 s(-1), with an 8-fold decrease in the burst of initial phosphorylation. With the S971A mutant, the rate constant decreases to 0.007 s(-1). PKAi(5-24) decreases the amount of the aspartylphosphate intermediate (EP) in Ccc2 wt by 50% within 1 min, but not in S258A, S971A, or S258A/S971A. The increase of the initial burst and the extremely slow phosphorylation when the "phosphomimetic" mutant S258D was assayed (k = 0.0036 s(-1)), indicate that electrostatic and conformational (non-electrostatic) mechanisms are involved in the regulatory role of Ser(258). Accumulation of an ADP-insensitive form in S971A demonstrates that Ser(971) is required to accelerate the hydrolysis of the E-P form during turnover. We propose that Ser(258) and Ser(971) are under long-range intramolecular, reciprocal and concerted control, in a sequential process that is crucial for catalysis and copper transport in the yeast copper ATPase.