Correction for Extraneous Background in X-Ray Microanalysis of Cell Cultures

Some practical aspects of the X-ray microanalysis of cell cultures have been investigated. Cells were cultured on titanium grids covered with Formvar films and analyzed at 100 kV either in the scanning transmission (STEM) or transmission mode (TEM) of the electron microscope. Different holders, grids and configurations were compared with respect to the relative contribution of different factors to the extraneous background in the X-ray spectrum. When low atomic number holders are used, the contribution to the spectrum of electrons scattered through high angles, may be negligible. In practice this may result in negative values for the contribution of these scattered electrons to the background. Computer programs for correction of the extraneous background should ignore these negative values and replace them by zero. When a brass holder is used, the contribution to the spectrum from electrons scattered through high angles becomes more important than that of the uncollimated radiation. The position of the analyzed cell relative to the grid bars is more important than the choice of grid or holder type. The data show that for the specimens used in the present study the correction for extraneous background is of little importance and can be neglected

Effect of Chronic Treatment with Diuretics on Mouse Liver: A Morphological and Microanalytical Investigation

In an attempt to produce an animal model for the disease cystic fibrosis (CF), mice were treated chronically with the diuretics amiloride and furosemide, in order to cause chronic inhibition of transepithelial ion transport. Experiments were carried out on adult mice (2 months treatment); in addition, pregnant mice were treated with diuretics, and tissue from offspring 2 and 7 days post partum was investigated. Since biliary cirrhosis is a common occurrence in CF, hepatocytes in the treated mice were investigated by X-ray microanalysis and by light and electron microscopy. Treatment with amiloride caused a significant decrease in cellular Na concentration in adult animals and in in utero treated mice 2 days after birth. The decrease in Na was parallelled by a decrease in Cl, but K levels were not affected. Furosemide caused a slight increase of cellular Na concentrations, especially in animals aged 7 days. In the adult animals, both amiloride and furosemide caused a significant decrease of the cellular Na and Cl levels. No signs of cirrhosis could be observed. Inconsistent changes in the accumulation of lipid droplets in hepatocytes of adult animals treated with amiloride were observed by electron microscopy. It can be concluded that chronic treatment with diuretics, even though it causes some, possibly pathological, changes of the liver, is only of very limited value for generating an animal model to study liver disease in CF

X-Ray Microanalysis of Epithelial and Secretory Cells in Culture

Cell cultures can be used to study ion transport processes. X-ray microanalysis of cell cultures at the cellular level gives interesting information that can complement electrophysiological and tracer studies. In this paper, methods for culturing and preparing a variety of epithelial and secretory cells (fibroblasts, insulinoma cells, bovine mammary epithelial cells, colon cancer cells) for X-ray microanalysis are presented. Results show that sometimes cell cultures are not homogeneous with respect to ion content or reaction to physiological stimuli. In colon cancer cell cultures, a high K and a low K cell subpopulation was found; these subpopulations also differed with respect to other elements. As examples of biological applications, chloride efflux was studied in fibroblasts and colon cancer cells, and strontium uptake in insulinoma cells. Chloride efflux from colon cancer cells is stimulated by cyclic AMP and vaso-active intestinal peptide (VIP), and can be inhibited by pretreatment of the cells with phorbol myristate acetate, which downregulates the cAMP-regulated chloride efflux mechanism

Diabetes, diabetes treatment, and mammographic density in Danish Diet, Cancer, and Health cohort

PURPOSE: We examined whether diabetes and diabetes treatment are associated with MD in a cohort study of Danish women above age of 50 years. METHODS: Study cohort consisted of 5,644 women (4,500 postmenopausal) who participated in the Danish Diet, Cancer, and Health cohort (1993–1997) and subsequently attended mammographic screening in Copenhagen (1993–2001). We used MD assessed at the first screening after the cohort entry, defined as mixed/dense or fatty. Diabetes diagnoses and diabetes treatments (diet, insulin, or oral antidiabetic agents) were self-reported at the time of recruitment (1993–1997). The association between MD and diabetes was analyzed by logistic regression adjusted for potential confounders. Effect modification by menopausal status and body mass index (BMI) was performed by introducing an interaction term into the model and tested by Wald test. RESULTS: Of 5,644 women with mean age of 56 years, 137 (2.4%) had diabetes and 3,180 (56.3%) had mixed/dense breasts. Having diabetes was significantly inversely associated with having mixed/dense breasts, in both, the crude model (odds ratio; 95% confidence interval: 0.33; 0.23–0.48), and after adjustment for adiposity and other risk factors (0.61; 0.40–0.92). Similar inverse associations were observed for 44 women who controlled diabetes by diet only and did not receive any medication (0.56; 0.27–1.14), and 62 who took oral antidiabetic agents only for diabetes (0.59; 0.32–1.09), while women taking insulin had increased odds of mixed/dense breasts (2.08; 0.68–6.35). There was no effect modification of these associations by menopausal status or BMI. CONCLUSIONS: Having diabetes controlled by diet or oral antidiabetic agents is associated with a decrease in MD, whereas taking insulin is associated with an increase in MD

SURFIN is a polymorphic antigen expressed on Plasmodium falciparum merozoites and infected erythrocytes

The surfaces of the infected erythrocyte (IE) and the merozoite, two developmental stages of malaria parasites, expose antigenic determinants to the host immune system. We report on surface-associated interspersed genes (surf genes), which encode a novel polymorphic protein family, SURFINs, present on both IEs and merozoites. A SURFIN expressed in 3D7 parasites, SURFIN4.2, was identified by mass spectrometric analysis of peptides cleaved off the surface of live IEs with trypsin. SURFINs are encoded by a family of 10 surf genes, including three predicted pseudogenes, located within or close to the subtelomeres of five of the chromosomes. SURFINs show structural and sequence similarities with exported surface-exposed proteins (PvSTP1, PkSICAvar, PvVIR, Pf332, and PfEMP1) of several Plasmodium species. SURFIN4.2 of a parasite other than 3D7 (FCR3S1.2) showed polymorphisms in the extracellular domain, suggesting sequence variability between genotypes. SURFIN4.2 not only was found cotransported with PfEMP1 and RIFIN to the IE surface, but also accumulated in the parasitophorous vacuole. In released merozoites, SURFIN4.2 was present in an amorphous cap at the parasite apex, where it may be involved in the invasion of erythrocytes. By exposing shared polymorphic antigens on IEs and merozoites, the parasite may coordinate the antigenic composition of these attachment surfaces during growth in the bloodstream

Cortical plasticity associated with stuttering therapy.

Abstract Neuroimaging studies have indicated that persistent developmental stuttering (PDS) may be associated both with an abnormality in white matter of left-hemispheric speech areas and a right-hemispheric hyperactivity. The latter may compensate for the deficient structural connectivity in the left hemisphere. To investigate the effects of stuttering therapy on brain activity nine male adults with PDS underwent functional magnetic resonance imaging (fMRI) before and within 12 weeks after fluency shaping therapy. Brain response differences during overt sentence reading before and after therapy were assessed by utilizing random effects analyses. After therapy, a more widespread activation was observed in frontal speech and language regions and temporal areas of both hemispheres, particularly and more pronounced on the left side. Interestingly, distinct posttreatment left-sided activation increases were located directly adjacent to a recently detected area of white matter anomaly [M. Sommer, M.A. Koch, W. Paulus, C. Weiller, C. Büchel (2002 Neumann et al. / Journal of Fluency Disorders 30 (2005) [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39] execution, and temporal areas. Hence, a therapeutic mechanism can be assumed to remodel brain circuitry close to the source of the dysfunction instead of reinforcing compensation via homologous contralateral brain networks. Educational objectives: The reader will learn about and be able to: (1) describe brain activation changes detected shortly after fluency-shaping therapy; (2) identify left-hemispheric regions where a (re)functionalization after fluency-shaping therapy seems to occur adjacent to a recently described abnormal white matter region in PDS subjects; and (3) discuss how an effective cerebral compensation mechanism for stuttering could work

Glycogen Synthase Kinase (GSK) 3β phosphorylates and protects nuclear myosin 1c from proteasome-mediated degradation to activate rDNA transcription in early G1 cells

Nuclear myosin 1c (NM1) mediates RNA polymerase I (pol I) transcription activation and cell cycle progression by facilitating PCAF-mediated H3K9 acetylation, but the molecular mechanism by which NM1 is regulated remains unclear. Here, we report that at early G1 the glycogen synthase kinase (GSK) 3β phosphorylates and stabilizes NM1, allowing for NM1 association with the chromatin. Genomic analysis by ChIP-Seq showed that this mechanism occurs on the rDNA as active GSK3β selectively occupies the gene. ChIP assays and transmission electron microscopy in GSK3β-/- mouse embryonic fibroblasts indicated that at G1 rRNA synthesis is suppressed due to decreased H3K9 acetylation leading to a chromatin state incompatible with transcription. We found that GSK3β directly phosphorylates the endogenous NM1 on a single serine residue (Ser-1020) located within the NM1 C-terminus. In G1 this phosphorylation event stabilizes NM1 and prevents NM1 polyubiquitination by the E3 ligase UBR5 and proteasome-mediated degradation. We conclude that GSK3β-mediated phosphorylation of NM1 is required for pol I transcription activation

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)