Characterization of the receptor for insulin-like growth factor on Leishmania promastigotes

Insulin-like growth factor (IGF)-I constitutively present in the skin is one of the first growth factors that Leishmania parasites encounter after transmission to the vertebrate host. We have previously shown that IGF-I is a potent growth-promoting factor for Leishmania parasites. IGF-I binds specifically to a single-site putative receptor at the parasite membrane, triggering a cascade of phosphorylation reactions. In the present article we characterize the receptor for IGF-I on Leishmania (Leishmania) mexicana promastigotes. The receptor is a monomeric glycoprotein with a molecular mass of 65 kDa and is antigenically related to the alpha chain of human type 1 IGF-I receptor. Upon IGF-I stimulation the receptor undergoes autophosphorylation on tyrosine residues with activation of its signaling pathway. Activation of the IGF-I receptor also leads to phosphorylation of an 185-kDa molecule that is homologous to the substrate of the insulin receptor present in human cells, the insulin receptor substrate 1 (IRS-1).     

Immobilization of lipase on chitin and its use in nonconventional biocatalysis

Chitin was functionalized with hexamethylenediamine followed by glutaraldehyde activation, and its capacity to bind Candida rugosa lipase was investigated. The loading of 250 units g(-1) support showed to be effective, resulting in a uniform enzyme fixation with high catalytic activity. Both free and immobilized lipases were characterized by determining the activity profile as a function of pH, temperature, and thermal stability. For the immobilized lipase, the influence of the reaction temperature and substrate polarity in nonconventional biocatalysis was also analyzed. Production of butyl esters was found to be dependent on the substrate partition coefficient, which accounts the greatest value for the system butanol and butyric acid. The highest enzyme activity was found for the system butanol and caprylic acid at a reaction temperature of 40 degrees C. Under such conditions, the operational stability tests indicated that a small enzyme deactivation occurs after 12 batches, revealing a biocatalyst half-life of 426.7 h.     

Dominant-negative p53-overexpression in skeletal muscle induces cell death and fiber atrophy in rats

The tumor suppressor p53 is thought to play a key role in the maintenance of cell size and homeostasis, but relatively little is known about its role in skeletal muscle. Based on its ability to suppress cell growth, we hypothesized that inhibiting the function of wild-type p53 through the overexpression of a dominant-negative p53 mutant (DDp53) could result in muscle fiber hypertrophy. To test this hypothesis, we electroporated adult rat tibialis anterior muscles with DDp53 and collected the tissue three weeks later. We confirmed successful overexpression of DDp53 on a histological and biochemical level and found pronounced changes to muscle architecture, metabolism, and molecular signaling. Muscle mass, fiber cross-sectional area, and fiber diameter significantly decreased with DDp53 overexpression. We found histopathological changes in DDp53 transfected muscle which were accompanied by increased levels of proteins that are associated with membrane damage and repair. In addition, DDp53 decreased oxidative phosphorylation complex I and V protein levels, and despite its negative effects on muscle mass and fiber size, caused an increase in muscle protein synthesis as assessed via the SUnSET technique. Interestingly, the increase in muscle protein synthesis was concomitant with a decrease in phospho-S6K1 (Thr389). Furthermore, the muscle wasting in the DDp53 electroporated leg was accompanied by a decrease in global protein ubiquitination and an increase in proteasome activity. In conclusion, overexpression of a dominant-negative p53 mutant in skeletal muscle results in decreased muscle mass, myofiber size, histological muscle damage, a metabolic phenotype, and perturbed homeostasis between muscle protein synthesis and degradation.Subject terms: Proteasome, Phosphorylation, Contractile proteins     

Performance bonuses and the quality of primary health care delivered by family health teams in Brazil: A difference-in-differences analysis

BackgroundPay-for-performance (P4P) programmes to incentivise health providers to improve quality of care have been widely implemented globally. Despite intuitive appeal, evidence on the effectiveness of P4P is mixed, potentially due to differences in how schemes are designed. We exploited municipality variation in the design features of Brazil’s National Programme for Improving Primary Care Access and Quality (PMAQ) to examine whether performance bonuses given to family health team workers were associated with changes in the quality of care and whether the size of bonus mattered.Methods and findingsFor this quasi-experimental study, we used a difference-in-differences approach combined with matching. We compared changes over time in the quality of care delivered by family health teams between (bonus) municipalities that chose to use some or all of the PMAQ money to provide performance-related bonuses to team workers with (nonbonus) municipalities that invested the funds using traditional input-based budgets. The primary outcome was the PMAQ score, a quality of care index on a scale of 0 to 100, based on several hundred indicators (ranging from 598 to 660) of health care delivery. We did one-to-one matching of bonus municipalities to nonbonus municipalities based on baseline demographic and economic characteristics. On the matched sample, we used ordinary least squares regression to estimate the association of any bonus and size of bonus with the prepost change over time (between November 2011 and October 2015) in the PMAQ score. We performed subgroup analyses with respect to the local area income of the family health team. The matched analytical sample comprised 2,346 municipalities (1,173 nonbonus municipalities; 1,173 bonus municipalities), containing 10,275 family health teams that participated in PMAQ from the outset. Bonus municipalities were associated with a 4.6 (95% CI: 2.7 to 6.4; p < 0.001) percentage point increase in the PMAQ score compared with nonbonus municipalities. The association with quality of care increased with the size of bonus: the largest bonus group saw an improvement of 8.2 percentage points (95% CI: 6.2 to 10.2; p < 0.001) compared with the control. The subgroup analysis showed that the observed improvement in performance was most pronounced in the poorest two-fifths of localities. The limitations of the study include the potential for bias from unmeasured time-varying confounding and the fact that the PMAQ score has not been validated as a measure of quality of care.ConclusionsPerformance bonuses to family health team workers compared with traditional input-based budgets were associated with an improvement in the quality of care.

Nasser Fardousi and colleagues investigate the association between performance bonuses and the quality of primary health care delivered by family health teams in Brazil.     

Plasmodium berghei leucine-rich repeat protein 1 downregulates protein phosphatase 1 activity and is required for efficient oocyst development

Protein phosphatase 1 (PP1) is a key enzyme for Plasmodium development. However, the detailed mechanisms underlying its regulation remain to be deciphered. Here, we report the functional characterization of the Plasmodium berghei leucine-rich repeat protein 1 (PbLRR1), an orthologue of SDS22, one of the most ancient and conserved PP1 interactors. Our study shows that PbLRR1 is expressed during intra-erythrocytic development of the parasite, and up to the zygote stage in mosquitoes. PbLRR1 can be found in complex with PbPP1 in both asexual and sexual stages and inhibits its phosphatase activity. Genetic analysis demonstrates that PbLRR1 depletion adversely affects the development of oocysts. PbLRR1 interactome analysis associated with phospho-proteomics studies identifies several novel putative PbLRR1/PbPP1 partners. Some of these partners have previously been characterized as essential for the parasite sexual development. Interestingly, and for the first time, Inhibitor 3 (I3), a well-known and direct interactant of Plasmodium PP1, was found to be drastically hypophosphorylated in PbLRR1-depleted parasites. These data, along with the detection of I3 with PP1 in the LRR1 interactome, strongly suggest that the phosphorylation status of PbI3 is under the control of the PP1–LRR1 complex and could contribute (in)directly to oocyst development. This study provides new insights into previously unrecognized PbPP1 fine regulation of Plasmodium oocyst development through its interaction with PbLRR1.     

Retinol (Vitamin A) Increases α-Synuclein, β-Amyloid Peptide,Tau Phosphorylation and RAGE Content in Human SH-SY5Y Neuronal Cell Line

Retinoids (vitamin A and derivatives) are recognized as essential factors for central nervous system (CNS) development. Retinol (vitamin A) also was postulated to be a major antioxidant component of diet as it modulates reactive species (RS) production and oxidative stress in biological systems. Oxidative stress plays a major role either in pathogenesis or development of neurodegenerative diseases, or even in both. Here we investigate the role of retinol supplementation to human neuron-derived SH-SY5Y cells over RS production and biochemical markers associated to neurodegenerative diseases expressed at neuronal level in Parkinson’s disease and Alzheimer’s disease: α-synuclein, β-amyloid peptide, tau phosphorylation and RAGE. Retinol treatment (24 h) impaired cell viability and increased intracellular RS production at the highest concentrations (7 up to 20 µM). Antioxidant co-treatment (Trolox 100 µM) rescued cell viability and inhibited RS production. Furthermore, retinol (10 µM) increased the levels of α-synuclein, tau phosphorylation at Ser396, β-amyloid peptide and RAGE. Co-treatment with antioxidant Trolox inhibited the increased in RAGE, but not the effect of retinol on α-synuclein, tau phosphorylation and β-amyloid peptide accumulation. These data indicate that increased availability of retinol to neurons at levels above the cellular physiological concentrations may induce deleterious effects through diverse mechanisms, which include oxidative stress but also include RS-independent modulation of proteins associated to progression of neuronal cell death during the course of neurodegenerative diseases.     

Distinct functions of maternal and somatic Pat1 protein paralogs

We previously identified Xenopus Pat1a (P100) as a member of the maternal CPEB RNP complex, whose components resemble those of P-(rocessing) bodies, and which is implicated in translational control in Xenopus oocytes. Database searches have identified Pat1a proteins in other vertebrates, as well as paralogous Pat1b proteins. Here we characterize Pat1 proteins, which have no readily discernable sequence features, in Xenopus oocytes, eggs, and early embryos and in human tissue culture cells. xPat1a and 1b have essentially mutually exclusive expression patterns in oogenesis and embryogenesis. xPat1a is degraded during meiotic maturation, via PEST-like regions, while xPat1b mRNA is translationally activated at GVBD by cytoplasmic polyadenylation. Pat1 proteins bind RNA in vitro, via a central domain, with a preference for G-rich sequences, including the NRAS 5′ UTR G-quadruplex-forming sequence. When tethered to reporter mRNA, both Pat proteins repress translation in oocytes. Indeed, both epitope-tagged proteins interact with the same components of the CPEB RNP complex, including CPEB, Xp54, eIF4E1b, Rap55B, and ePAB. However, examining endogenous protein interactions, we find that in oocytes only xPat1a is a bona fide component of the CPEB RNP, and that xPat1b resides in a separate large complex. In tissue culture cells, hPat1b localizes to P-bodies, while mPat1a-GFP is either found weakly in P-bodies or disperses P-bodies in a dominant-negative fashion. Altogether we conclude that Pat1a and Pat1b proteins have distinct functions, mediated in separate complexes. Pat1a is a translational repressor in oocytes in a CPEB-containing complex, and Pat1b is a component of P-bodies in somatic cells.     

Visual Evoked Cortical Potential (VECP) Elicited by Sinusoidal Gratings Controlled by Pseudo-Random Stimulation

The contributions of contrast detection mechanisms to the visual cortical evoked potential (VECP) have been investigated studying the contrast-response and spatial frequency-response functions. Previously, the use of m-sequences for stimulus control has been almost restricted to multifocal electrophysiology stimulation and, in some aspects, it substantially differs from conventional VECPs. Single stimulation with spatial contrast temporally controlled by m-sequences has not been extensively tested or compared to multifocal techniques. Our purpose was to evaluate the influence of spatial frequency and contrast of sinusoidal gratings on the VECP elicited by pseudo-random stimulation. Nine normal subjects were stimulated by achromatic sinusoidal gratings driven by pseudo random binary m-sequence at seven spatial frequencies (0.4–10 cpd) and three stimulus sizes (4°, 8°, and 16° of visual angle). At 8° subtence, six contrast levels were used (3.12–99%). The first order kernel (K1) did not provide a consistent measurable signal across spatial frequencies and contrasts that were tested–signal was very small or absent–while the second order kernel first (K2.1) and second (K2.2) slices exhibited reliable responses for the stimulus range. The main differences between results obtained with the K2.1 and K2.2 were in the contrast gain as measured in the amplitude versus contrast and amplitude versus spatial frequency functions. The results indicated that K2.1 was dominated by M-pathway, but for some stimulus condition some P-pathway contribution could be found, while the second slice reflected the P-pathway contribution. The present work extended previous findings of the visual pathways contribution to VECP elicited by pseudorandom stimulation for a wider range of spatial frequencies.     

Imatinib mesylate affects extracellular ATP catabolism and expression of NTPDases in a chronic myeloid leukemia cell line

Purinergic Signalling - Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm, characterized by the occurrence of the t(9;22)(q34;q11) translocation. First-line therapy for CML consists...