Regulation of Plasmodium falciparum Glideosome Associated Protein 45 (PfGAP45) Phosphorylation

The actomyosin motor complex of the glideosome provides the force needed by apicomplexan parasites such as Toxoplasma gondii (Tg) and Plasmodium falciparum (Pf) to invade their host cells and for gliding motility of their motile forms. Glideosome Associated Protein 45 (PfGAP45) is an essential component of the glideosome complex as it facilitates anchoring and effective functioning of the motor. Dissection of events that regulate PfGAP45 may provide insights into how the motor and the glideosome operate. We found that PfGAP45 is phosphorylated in response to Phospholipase C (PLC) and calcium signaling. It is phosphorylated by P. falciparum kinases Protein Kinase B (PfPKB) and Calcium Dependent Protein Kinase 1 (PfCDPK1), which are calcium dependent enzymes, at S89, S103 and S149. The Phospholipase C pathway influenced the phosphorylation of S103 and S149. The phosphorylation of PfGAP45 at these sites is differentially regulated during parasite development. The localization of PfGAP45 and its association may be independent of the phosphorylation of these sites. PfGAP45 regulation in response to calcium fits in well with the previously described role of calcium in host cell invasion by malaria parasite

Grape Seed Proanthocyanidins Inhibit the Invasiveness of Human HNSCC Cells by Targeting EGFR and Reversing the Epithelial-To-Mesenchymal Transition

Head and neck squamous cell carcinoma (HNSCC) is responsible for approximately 20,000 deaths per year in the United States. Most of the deaths are due to the metastases. To develop more effective strategies for the prevention of metastasis of HNSCC cells, we have determined the effect of grape seed proanthocyanidins (GSPs) on the invasive potential of HNSCC cell and the mechanisms underlying these effects using OSC19 cells as an in vitro model. Using cell invasion assays, we established that treatment of the OSC19 cells with GSPs resulted in a dose-dependent inhibition of cell invasion. EGFR is over-expressed in 90% of HNSCCs and the EGFR inhibitors, erlotinib and gefitinib, are being explored as therapies for this disease. We found that GSPs treatment reduced the levels of expression of EGFR in the OSC19 cells as well as reducing the activation of NF-κB/p65, a downstream target of EGFR, and the expression of NF-κB-responsive proteins. GSPs treatment also reduced the activity of ERK1/2, an upstream regulator of NF-κB and treatment of the cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, inhibited cell invasion. Overexpression of EGFR and high NF-κB activity play a key role in the epithelial-to-mesenchymal transition, which is of critical importance in the processes underlying metastasis, and we found treatment with GSPs enhanced the levels of epithelial (E-cadherin, cytokeratins and desmoglein-2) and reduced the levels of mesenchymal (vimentin, fibronectin, N-cadherin and Slug) biomarkers in the OSC19 cells. These results indicate that GSPs have the ability to inhibit HNSCC cell invasion, and do so by targeting the expression of EGFR and activation of NF-κB as well as inhibiting the epithelial-to-mesenchymal transition

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

Meeting abstrac

NADPH-diaphorase reactive pyramidal neurons in Ammon's horn and the subiculum of the rat hippocampal formation.

NADPH-diaphorase histochemistry has been shown to stain cells which contain nitric oxide synthase, an enzyme responsible for the biosynthesis of the freely diffusable gas nitric oxide. A number of studies have mapped the distribution of NADPH-diaphorase-reactive neurons in the hippocampal formation but they have failed to yield consistent data. The major point of controversy concerns the presence of NADPH-diaphorase-reactive pyramidal cells in the CA1 subfield of the rat hippocampal formation. The present results show that CA1 pyramidal neurons do contain nitric oxide synthase (NOS) which can be reliably demonstrated with the appropriate histochemical procedure. One of the critical determinants of CA1 pyramidal cell NADPH-diaphorase activity is shown to be incubation of brains in sucrose solution prior to histochemical processing. Subicular pyramidal cells were also found to contain NOS and to possess NADPH-diaphorase activity. These results explain a number of contradictory reports in the literature relating to the presence of NADPH-diaphorase activity in hippocampal principal cells. Additionally, densitometric analysis carried out on 20 microns thick sections, from brains incubated in sucrose solution, indicated that there were characteristic gradients. The intensity of NADPH-diaphorase activity in pyramidal cells located in the ventral subiculum was found to be greater than those in the dorsal subiculum. A similar, yet marginal, trend was apparent for pyramidal cells in CA1 and CA3, as well as nonpyramidal cells in CA1. At both dorsal and ventral levels, NADPH-diaphorase-positive subicular pyramidal cells and CA1 nonpyramidal cells also show a greater intensity than CA1 or CA3 reactive pyramidal neurons. This study also showed that tissue incubation in sucrose solution prior to immunocytochemistry, enhanced immunoreactivity of the endothelial isoform of NOS whilst having little effect on neuronal NOS reactivity

A comparison of the density of NADPH-diaphorase-reactive neurons in the fascia dentata and Ammon's horn between 6-month and 12-month old dark agouti rats.

The present study aimed to assess the developmental progress of the hippocampal nitric oxide (NO) system within adulthood by comparing the density of NO-producing neurons in the fascia dentata and Ammon's horn in two groups of adult male rats using NADPH-diaphorase (NADPH-d) histochemistry. One group comprised 6-month-old rats (early adulthood), and the other 12-month-old rats (middle-adulthood). Areal density (number of neurons per unit area) of NADPH-d positive neurons along the three hippocampal axes (septo-temporal, transverse and radial axes) were subjected to quantitative analyses. There were significant variations in the density of NADPH-d-reactive neurons along the transverse and radial axes of the hippocampus, similar to what have been described previously. Comparison between 6-month and 12-month-old rats indicated a substantial reduction in the density of NADPH-d-reactive neurons in the fascia dentata (69%) and Ammon's horn (54%) of the latter group. This reduction was relatively uniform along the septotemporal and radial axes, but appeared to be more pronounced in the fascia dentata and in the proximal region of Ammon's horn. Our finding showed that the hippocampal NO system can undergo significant changes within adulthood. It further highlighted the possibility that an age-related reduction in the capacity to produce NO may not be directly responsible for the cognitive decline associated with senescence, but rather predisposes neuronal degeneration in later life

Neonatal nonhandling and in utero prenatal stress reduce the density of NADPH-diaphorase-reactive neurons in the fascia dentata and Ammon's horn of rats.

The density of nitric oxide (NO)-producing neurons in the fascia dentata and Ammon's horn was assessed in 6-month-old male rats using NADPH-diaphorase (NADPH-d) histochemistry. Two separate experiments investigated whether (1) the complete absence of neonatal handling or (2) the administration of periodic prenatal stress could affect the expression and distribution of NADPH-d reactivity in the hippocampus, when compared with rats raised in normal standard laboratory conditions. Experiment 1 demonstrated that adult rats that received no handling during neonatal development (from birth to postnatal day 22) showed a very substantial reduction in NADPH-d-positive neurons per unit area throughout the entire hippocampus when compared with rats that received regular daily handling in this period. Quantitative analysis further revealed that this effect was significantly more pronounced in Ammon's horn than in the fascia dentata, and within Ammon's horn the dorsal region was selectively more affected. Experiment 2 showed that prenatal stress, which involved the administration of daily restraint stress to pregnant dams throughout the gestation period, also led to a reduction in NADPH-d reactivity in the hippocampus of the offspring of these dam when they reached adulthood. The present results suggest that behavioral manipulations in the early neonatal or prenatal period can significantly alter the neurodevelopment of the hippocampal NO system and these changes might be related to some of the behavioral abnormalities that emerge later in adulthood

Temporal trends in severe malaria in Chittagong, Bangladesh.

BACKGROUND: Epidemiological data on malaria in Bangladesh are sparse, particularly on severe and fatal malaria. This hampers the allocation of healthcare provision in this resource-poor setting. Over 85% of the estimated 150,000-250,000 annual malaria cases in Bangladesh occur in Chittagong Division with 80% in the Chittagong Hill Tracts (CHT). Chittagong Medical College Hospital (CMCH) is the major tertiary referral hospital for severe malaria in Chittagong Division. METHODS: Malaria screening data from 22,785 inpatients in CMCH from 1999-2011 were analysed to investigate the patterns of referral, temporal trends and geographical distribution of severe malaria in Chittagong Division, Bangladesh. RESULTS: From 1999 till 2011, 2,394 malaria cases were admitted, of which 96% harboured Plasmodium falciparum and 4% Plasmodium vivax. Infection was commonest in males (67%) between 15 and 34 years of age. Seasonality of malaria incidence was marked with a single peak in P. falciparum transmission from June to August coinciding with peak rainfall, whereas P. vivax showed an additional peak in February-March possibly representing relapse infections. Since 2007 there has been a substantial decrease in the absolute number of admitted malaria cases. Case fatality in severe malaria was 18% from 2008-2011, remaining steady during this period.A travel history obtained in 226 malaria patients revealed only 33% had been to the CHT in the preceding three weeks. Of all admitted malaria patients, only 9% lived in the CHT, and none in the more remote malaria endemic regions near the Indian border. CONCLUSIONS: The overall decline in admitted malaria cases to CMCH suggests recent control measures are successful. However, there are no reliable data on the incidence of severe malaria in the CHT, the most endemic area of Bangladesh, and most of these patients do not reach tertiary health facilities. Improvement of early treatment and simple supportive care for severe malaria in remote areas and implementation of a referral system for cases requiring additional supportive care could be important contributors to further reducing malaria-attributable disease and death in Bangladesh

Filamin A mutations cause periventricular heterotopia with Ehlers-Danlos syndrome

Objective: To define the clinical, radiologic, and genetic features of periventricular heterotopia (PH) with Ehlers-Danlos syndrome (EDS). Methods: Exonic sequencing and single stranded conformational polymorphism (SSCP) analysis was performed on affected individuals. Linkage analysis using microsatellite markers on the X-chromosome was performed on a single pedigree. Western blotting evaluated for loss of filamin A ( FLNA) protein and Southern blotting assessed for any potential chromosome rearrangement in this region. Results: The authors report two familial cases and nine additional sporadic cases of the EDS-variant form of PH, which is characterized by nodular brain heterotopia, joint hypermobility, and development of aortic dilatation in early adulthood. MRI typically demonstrated bilateral nodular PH, indistinguishable from PH due to FLNA mutations. Exonic sequencing or SSCP analyses of FLNA revealed a 2762 delG single base pair deletion in one affected female. Another affected female harbored a C116 single point mutation, resulting in an A39G change. A third affected female had a 4147 delG single base pair deletion. One pedigree with no detectable exonic mutation demonstrated positive linkage to the FLNA locus Xq28, an affected individual in this family also had no detectable FLNA protein, but no chromosomal rearrangement was detected. Conclusion: These results suggest that the Ehlers-Danlos variant of periventricular heterotopia ( PH), in part, represents an overlapping syndrome with X-linked dominant PH due to filamin A mutations