Increase In Relative Level Research Articles

Abstract 3040: Quantitative Proteomic Analysis Of HDL Shows Apolipoprotein M (ApoM) As A Possible Marker For COVID-19 Mortality

Introduction: High-density lipoprotein (HDL) comprises a heterogeneous group of particles that differ in many aspects, such as density, size, composition and function. The use of mass spectrometry-based proteomics to investigate HDL protein composition has brought some new insights into the field. The qualitative and quantitative HDL proteome is remodeled in different disease states, such as diabetes mellitus, hypercholesterolemia and kidney dysfunction. Beyond its established role in inflammatory and cardiovascular diseases, HDL's involvement in infectious diseases, such as COVID-19, has recently come to light. Here, we investigate the remodeling of HDL proteome in hospitalized and non-hospitalized COVID-19 patients. Hypothesis: We hypothesize HDL proteome is remodeled in infectious diseases and may be a marker and perhaps a predictor of a poor prognosis. Methods: We implemented a robust, global label-free quantification proteomic approach to unravel relative changes in HDL protein composition. We analyzed the proteome of COVID-19 subjects from Brazil categorized in two groups, hospitalized (n=30) and non-hospitalized (n=11). Results: Comparative analysis between hospitalized and non-hospitalized patients revealed a more than 50% increase in relative levels of certain proteins in hospitalized patients, including the inflammatory proteins serum amyloid A1 and A2, as well as pulmonary surfactant-associated protein B. We also observed reductions in the lipid-metabolism related apolipoprotein A2 and phospholipid transfer protein. These findings, independent of sex, triglyceride and HDL-cholesterol, suggest an inflammatory remodeling of the HDL particles. Further analysis of surviving (n=35) and deceased patients (n=6) indicated a negative association between ApoM levels and odds of death due to COVID-19 complications (odds ratio per 1-SD increase in ApoM was 0.27 [0.07 to 0.72], P=0.007). Conclusions: Our results point out HDL remodeling is a marker of COVID-19 severity and ApoM-carried sphingosine-1-phosphate signaling pathway may play a pivotal role in the disease outcome.

High stability of blood parameters during mouse lifespan: sex-specific effects of every-other-day fasting.

Every-other-day fasting (EODF) is one type of caloric restriction that is proposed to have significant health benefits, including slowing aging-related processes. The present study evaluated multiple parameters of blood homeostasis comparing mice of different ages and mice on different diet regimes: ad libitum (AL) versus EODF. Hematological and classical biochemical parameters of blood were measured in young (6-month), middle-aged (12-month) and old (18-month) C57BL/6J mice of both sexes subjected either to EODF, or AL feeding. Middle-aged AL males showed a decrease in erythrocyte and total leucocyte counts and an increase in plasma alkaline phosphatase activity, whereas old animals showed a decrease in relative levels of lymphocytes and an increase in relative levels of neutrophils, a decrease in plasma lactate and an increase in total cholesterol levels, compared to young mice. AL-fed females demonstrated higher stability of blood parameters during aging than malesdid. The EODF regimen did not significantly affect hematological parameters in females but prevented a decline in total leukocyte count with age in males. In both sexes, EODF partially prevented age-associated changes in levels of plasma lactate and cholesterol and activity of alkaline phosphatase. Thus, during normal aging, mice showed a sex-dependent maintenance of blood homeostasis which was not significantly affected by EODF.

The genetic consequences of dog breed formation-Accumulation of deleterious genetic variation and fixation of mutations associated with myxomatous mitral valve disease in cavalier King Charles spaniels.

Selective breeding for desirable traits in strictly controlled populations has generated an extraordinary diversity in canine morphology and behaviour, but has also led to loss of genetic variation and random entrapment of disease alleles. As a consequence, specific diseases are now prevalent in certain breeds, but whether the recent breeding practice led to an overall increase in genetic load remains unclear. Here we generate whole genome sequencing (WGS) data from 20 dogs per breed from eight breeds and document a ~10% rise in the number of derived alleles per genome at evolutionarily conserved sites in the heavily bottlenecked cavalier King Charles spaniel breed (cKCs) relative to in most breeds studied here. Our finding represents the first clear indication of a relative increase in levels of deleterious genetic variation in a specific breed, arguing that recent breeding practices probably were associated with an accumulation of genetic load in dogs. We then use the WGS data to identify candidate risk alleles for the most common cause for veterinary care in cKCs-the heart disease myxomatous mitral valve disease (MMVD). We verify a potential link to MMVD for candidate variants near the heart specific NEBL gene in a dachshund population and show that two of the NEBL candidate variants have regulatory potential in heart-derived cell lines and are associated with reduced NEBL isoform nebulette expression in papillary muscle (but not in mitral valve, nor in left ventricular wall). Alleles linked to reduced nebulette expression may hence predispose cKCs and other breeds to MMVD via loss of papillary muscle integrity.

Consumer awareness of the use of food labels in Lagos state, Nigeria

The increase in relative level of education and the growing middle-class income earners in Nigeria have made packaged food items attractive to consumers. These foods provide handy, nutritious and convenient food for consumers; however, they also come with public health concerns. From a policy perspective, food labelling can gauge consumers’ response to the safety conditions of packaged food. However, understanding consumers’ awareness and the use of food label information has attracted little research attention in many developing countries in sub-Saharan Africa. In this study, we investigate consumers’ awareness of the use of food safety labels in Lagos state, Nigeria. We used primary data collected using pretested structured questionnaire from 220 food shoppers who bought pre-packaged foods . A two-stage sampling technique was used to select the respondents. Consumers’ awareness and use of food safety information was revealed using descriptive statistics ; Ordinal Regression Analysis was used to examine the socioeconomic factors determining the frequency of reading food safety labels . The study therefore recommends that there should be continuous awareness campaigns on the importance of food labels in ensuring safety and food producers should make sure their food labels are legible.

Consumer awareness of the use of food labels in Lagos state, Nigeria

The increase in relative level of education and the growing middle-class income earners in Nigeria have made packaged food items attractive to consumers. These foods provide handy, nutritious and convenient food for consumers; however, they also come with public health concerns. From a policy perspective, food labelling can gauge consumers’ response to the safety conditions of packaged food. However, understanding consumers’ awareness and the use of food label information has attracted little research attention in many developing countries in sub-Saharan Africa. In this study, we investigate consumers’ awareness of the use of food safety labels in Lagos state, Nigeria. We used primary data collected using pretested structured questionnaire from 220 food shoppers who bought pre-packaged foods. A two-stage sampling technique was used to select the respondents. Consumers’ awareness and use of food safety information was revealed using descriptive statistics; Ordinal Regression Analysis was used to examine the socioeconomic factors determining the frequency of reading food safety labels. The study therefore recommends that there should be continuous awareness campaigns on the importance of food labels in ensuring safety and food producers should make sure their food labels are legible.

Acute and subacute toxicity of the drug based on tylosin tartrate


 The article presents the results of a study of the acute and subacute toxicity of the drug TILOTAR (a water-soluble powder for oral administration) made on the basis of tilmicosin. As a result of the research it was found that the drug “TILOTAR” refers to the 4th class of toxicity, that is, to low-toxic substances. After intragastric administration of the drug to white mice and rats at doses of 2500, 5000, 7500 mg/kg, all animals remained alive. Short-term inhibition was recorded in laboratory animals, which were given the drug at the maximum dose. Further changes in the clinical state of the animals of the experimental groups were not observed. The study of subacute toxicity of the drug “TILOTAR” showed that 14-day administration of a therapeutic dose of the drug leads to a decrease in erythropoiesis, the appearance of old forms of erythrocytes, damage to hepatocytes, the appearance of eosinophils, γ-globulins, a decrease in protein biosynthesis, increased kidney function. The introduction of a 10-fold dose within 14 days reduced the body weight of the rats, leading to abnormalities in the liver and kidneys, and a decrease in the mass of the spleen. An increase in urea blood concentration and a decrease in creatinine may indicate impaired renal function in animals that were given a tenfold dose of the drug, and an increase in the level of α- and γ-globulins against the background of a decrease in body weight of rats indicates an overload of the body’s immune system. In rats of the therapeutic group, homeostasis was maintained by enhancing kidney function, accompanied by a decrease in the number of erythrocytes due to young forms and the appearance of mature erythrocytes with a high level of hemoglobin (MCH), some destruction of hepatocytes (high level of alkaline phosphatase activity) while reducing the amount of albumin, an increase in relative levels of α- and γ-globulins. In the conducted experiments it was established that the macroscopic and microscopic structure of the internal organs of rats, while studying the subacute toxicity of the drug “TILOTAR”, was preserved, no macroscopic changes were revealed. In rats receiving a 10-fold therapeutic dose of the drug for 14 days, focal protein dystrophy of the liver and kidneys was histologically established.

Abstract P2-06-17: A novel interaction of AURKA with MAPK pathway in breast cancer cells as a potential therapeutic target

Abstract Background: Aurora A (AURKA) is a mitotic kinase responsible for centrosome segregation and mitotic spindle formation. In normal cells, expression of AURKA is highly regulated and is predominantly restricted to G2/M phases of the cell cycle. Unlike healthy cells, cancer cells overexpress AURKA through all phases of the cell cycle resulting in the acquisition of alternate non-mitotic functions. Little is known about cellular functions regulated by AURKA and its interaction with other signaling molecules. Here, we report a novel interaction between AURKA and the mitogen-activated protein kinase (MAPK) pathway at the level of MEK1 in breast cancer cells. This interaction may serve as a novel target as well as demonstrate by an additive cytotoxic effect of AURKA- and MEK1/2-specific inhibitors against estrogen positive (ER+) and triple negative breast cancer (TNBC) cells. Results: We show that treatment of ER+ HER2- MCF-7, ER- HER2+ SKBR3 and ER- HER2- BT549 cells with AURKA specific inhibitors alisertib, MK8745 and Aurora A Inhibitor I resulted in over 2-fold increase in relative levels of poMEK1/2 and poERK1/2 compared to untreated controls. The activation of the MAPK pathway was rapid with changes seen within 5 min after treatment with AURKA inhibitors and was sustained for at least 48 hours. Treatment with the pan RAF inhibitor TAK-632 did not diminish alisertib-induced poERK and poMEK1/2. Alternatively, treatment with the MEK1/2 specific inhibitor PD0325901 completely abrogated alisertib-induced phosphorylation of MEK1/2 and ERK1/2. In situ proximity ligation and pull down assays demonstrated AURKA and MEK1/2 direct interaction. In vitro kinase assay showed direct phosphorylation of MEK1 by AURKA. Combined treatment of alisertib and PD0325901 in vitro revealed significant additive cytotoxic effect in MCF-7 and BT549 cells when compared to either agent used alone (p< 0.008 and p<0.011; p <0.04 and p<0.028) with early trend toward significance in survival in a BT549 xenograft breast cancer in vivo model. Conclusions: Our data shows a novel AURKA-MEK1 interaction in breast cancer cells. In depth in vivo analysis is ongoing. The results reveal a promising new strategy for the treatment TNBC patients using a combination of AURKA and MEK1/2 inhibitors. Citation Format: Gandhi S, Gil M, Khoury T, Takabe K, Puzanov I, Gelman I, D'Assoro A, Opyrchal M. A novel interaction of AURKA with MAPK pathway in breast cancer cells as a potential therapeutic target [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-17.

Association of Estrogen Metabolism with Breast Cancer Risk in Different Cohorts of Postmenopausal Women.

Endogenous estradiol and estrone are linked causally to increased risks of breast cancer. In this study, we evaluated multiple competing hypotheses for how metabolism of these parent estrogens may influence risk. Prediagnostic concentrations of estradiol, estrone, and 13 metabolites were measured in 1,298 postmenopausal cases of breast cancer and 1,524 matched controls in four separate patient cohorts. The median time between sample collection and diagnosis was 4.4 to 12.7 years across the cohorts. Estrogen analytes were measured in serum or urine by liquid chromatography-tandem mass spectrometry. Total estrogen levels (summing all 15 estrogens/estrogen metabolites) were associated strongly and positively with breast cancer risk. Normalizing total estrogen levels, we also found that a relative increase in levels of 2-hydroxylation pathway metabolites, or in the ratio of 2-hydroxylation:16-hydroxylation pathway metabolites, were associated inversely with breast cancer risk. These associations varied by total estrogen levels, with the largest risk reductions occurring in women in the highest tertile. With appropriate validation, these findings suggest opportunities for breast cancer prevention by modifying individual estrogen metabolism profiles through either lifestyle alterations or chemopreventive strategies. Cancer Res; 77(4); 918-25. ©2017 AACR.

LPS mediated decreases in immune cells recruitment on or near lymphatics impairs lymphatic contractility

Elevated levels of dietary endotoxins like lipopolysaccharide (LPS) are linked to several chronic inflammatory pathologies involving the lymphatics. However, the mechanisms of LPS modulated changes in lymphatic contractility have not been understood. Our hypothesis is that LPS mediated inflammation modulates the immune cell populations on or in vicinity of the lymphatics, resulting in changes in cytokines and chemokines that impair lymphatic function. To demonstrate this, rats were injected with LPS, and immune cells (eosinophils, neutrophils, macrophages and monocytes) were detected by confocal microscopy after 6hr, 24hr and 3d in whole mount mesenteric lymphatic preparations. We found a significant reduction in eosinophils and neutrophils 3d post LPS treatment and an increase in macrophages and monocytes. Treatment of lymphatic muscle cells with some of the key cytokines released by these cells (IL4, IL13, IL5 and IL8) showed an increase in relative levels of myosin light chain 20 phosphorylation by activation of AKT and ERK1/2. These cytokines also mitigated LPS induced impairment of lymphatic contractility in a time‐dependent manner. Together, our data show that LPS impairs lymphatic function by decreasing neutrophils and eosinophils on/near lymphatic vessels, thereby reducing the availability of key cytokines and their effects on lymphatic muscle. AHA 09POST2280005 to SC; KO2 HL86650 to MM

The Erythropoiesis Stimulating Agent Omontys/Peginesatide up-Modulates Erythroid Progenitor Formation, and Cell Surface Erythropoietin Receptor Levels.

AbstractAbstract 2089Peginesatide is a peptide-based erythropoietin receptor (EPOR) agonist with recent FDA approval for treating the anemia of chronic kidney disease among adult dialysis patients. Although peginesatide exhibits a 47.9h half-life when given IV in dialysis patients, it is administered once-monthly. Taken together, this predicts that additional functional properties contribute to peginesatide’s durable erythropoiesis stimulating activity. Here we report on three such properties. During ex vivo murine bone marrow erythroid development, compared directly to rHuEPO, peginesatide first enhanced KitposCD71pos progenitor cell expansion (including KitposCD71lowCD36posCD13pos erythromyelo- progenitors). Second, peginesatide exhibited a 1300 minute EPOR residence time vs. 77 minutes for rHuEPO. Third, the culture of EPO-dependent human UT7epo cells in peginesatide led to substantial EPOR up-modulation, as well as an apparent lessening of the processing of mature EPOR’s. Furthermore, in studies that compared peginesatide vs. rHuEPO effects on the erythroid development of human bone marrow-derived CD34pos progenitors, the following differences were observed. At days 6–10 of culture, peginesatide gave rise to increased frequencies (up to 200% as compared to rHuEPO) of KitposCD71pos co-positive erythroid progenitors. At early stages (d2-d4), cell-surface EPOR levels also were elevated among progenitors expanded in peginesatide. When levels of CD13posCD36pos co-positive cells were analyzed, frequencies of these erythromyelo-progenitors also were heightened up to three-fold. Analyses of cultures at later time-points (eg, d10 of culture) indicated that peginesatide and rHuEPO supported the formation of GPAhigh erythroblasts at similarly high frequencies. In addition, cytospin analyses revealed morphological distinctions for multicellular proerythroblast complexes formed in peginesatide. Peginesatide’s persistent erythropoietic activity allowing for once-monthly dosing therefore is likely to involve novel effects on erythromyelo-progenitor recruitment, and increased EPOR cell surface expression including apparent increases in relative levels of full-length EPOR forms. Disclosures:Green:Affymax, Inc.: Employment. Leu:Affymax, Inc.: Employment. Mortensen:Affymax, Inc.: Employment. Young:Affymax, Inc.: Prior employment Other. Schatz:Affymax, Inc.: Employment. Wojchowski:Affymax, Inc.: Membership on an entity's Board of Directors or advisory committees.

Acute administration of ketamine in rats increases hippocampal BDNF and mTOR levels during forced swimming test

Introduction. Previous studies have shown that a single sub-anesthetic dose of ketamine exerts fast-acting antidepressant effects in patients and in animal models of depression. However, the underlying mechanisms are not totally understood. This study aims to investigate the effects of acute administration of different doses of ketamine on the immobility time of rats in the forced swimming test (FST) and to determine levels of hippocampal brain-derived neurotrophic factor (BDNF) and mammalian target of rapamycin (mTOR).Methods. Forty male Wistar rats weighing 180–220 g were randomly divided into four groups (n = 10 each): group saline and groups ketamine 5, 10, and 15 mg/kg. On the first day, all animals were forced to swim for 15 min. On the second day ketamine (5, 10, and 15 mg/kg, respectively) was given intraperitoneally, at 30 min before the second episode of the forced swimming test. Immobility times of the rats during the forced swimming test were recorded. The animals were then decapitated. The hippocampus was harvested for determination of BDNF and mTOR levels.Results. Compared with group saline, administration of ketamine at a dose of 5, 10, and 15 mg/kg decreased the duration of immobility (P < 0.05 for all doses). Ketamine at doses of both 10 and 15 mg/kg showed a significant increase in the expression of hippocampal BDNF (P < 0.05 for both doses). Ketamine given at doses of 5, 10, and 15 mg/kg showed significant increases in relative levels of hippocampal p-mTOR (P < 0.05 for all doses)Conclusion. The antidepressant effect of ketamine might be related to the increased expression of BDNF and mTOR in the hippocampus of rats.

Effect of pine pollen on kidney mitochondria DNA deletion mutation in senile mice

To study the effect of pine pollen on Kidney Mitochondria DNA Deletion Mutation (mtDNA) in senile mice. Kunming senile mice were randomly divided into the pine pollen group, and the senile control group. And a young control group was randomly selected. Mouse in the pine pollen group were orally administered with pine pollen (750 mg x kg(-1)) daily. The young control group and the senile control group were orally administered with isometric 0.9% sodium chloride injection. After 60 days, deletion mutation of mtDNA were detected by PCR technology and photodensity scan. Relative level of MDA and activity of SOD in kidney tissues were detected. The senile control group showed significant increase in relative level and deletion mutation of mtDNA (P < 0.05). Compared with the senile control group, the pine pollen group showed decreased depletion of kidney mtDNA (P < 0.05). Pine pollen can decrease MDA volume and increase the activity of SOD significantly (P < 0.05). Pine pollen can inhibit deletion mutation of mtDNA in senile mice, suggesting that pine pollen can reduce oxidative damage of mtDNA and protect mtDNA. Accordingly, it provides a possible mechanism of anti-aging effect of pine pollen at the molecular level.

PMO-048 Reduction in pro-inflammatory cytokines after weight loss surgery: a prospective study

IntroductionMorbid obesity is associated with a pro-inflammatory state, reflected by a relative increase in levels of pro-inflammatory cytokines and corresponding decrease in anti-inflammatory cytokines. The production of active mediators by...

Human Plasma Glycome in Attention-Deficit Hyperactivity Disorder and Autism Spectrum Disorders

Over a half of all proteins are glycosylated, and their proper glycosylation is essential for normal function. Unfortunately, because of structural complexity of nonlinear branched glycans and the absence of genetic template for their synthesis, the knowledge about glycans is lagging significantly behind the knowledge about proteins or DNA. Using a recently developed quantitative high throughput glycan analysis method we quantified components of the plasma N-glycome in 99 children with attention-deficit hyperactivity disorder (ADHD), 81 child and 5 adults with autism spectrum disorder, and a total of 340 matching healthy controls. No changes in plasma glycome were found to associate with autism spectrum disorder, but several highly significant associations were observed with ADHD. Further structural analysis of plasma glycans revealed that ADHD is associated with increased antennary fucosylation of biantennary glycans and decreased levels of some complex glycans with three or four antennas. The design of this study prevented any functional conclusions about the observed associations, but specific differences in glycosylation appears to be strongly associated with ADHD and warrants further studies in this direction.

Annexin A2 Mediates Up-regulation of NF-κB, β-catenin, and Stem Cell in Response to Progastrin in Mice and HEK-293 Cells

Prograstrin induces proliferation in colon crypts by activating p65nuclear factor-κB (NF-κB) (p65) and β-catenin. We investigated whether Annexin A2 (AnxA2), a progastrin receptor, activates NF-κB and β-catenin in vivo. ANXA2-null (ANXA2(-/-)) and wild-type (ANXA2(+/+)) mice were studied, along with clones of progastrin-responsive HEK-293 cells that stably expressed full-length progastrin (HEK-mGAS) or an empty vector (HEK-C). Small interfering RNA was used to down-regulate AnxA2, p65NF-κB, and β-catenin in cells. Proliferation and activation of p65 and β-catenin increased significantly in HEK-mGAS compared with HEK-C clones. HEK-mGAS cells had a 2- to 4-fold increase in relative levels of c-Myc, cyclooxygenase (COX)-2, CyclinD1, double cortin CAM kinase-like 1 (DCAMKL+1), and CD44, compared with HEK-C clones. Down-regulation of AnxA2 in HEK-mGAS clones reduced activation of NF-κB and β-catenin, as well as levels of DCAMKL+1. Surprisingly, down-regulation of β-catenin had no effect on activation of p65NF-κB, whereas down-regulation of p65 significantly reduced activation of β-catenin in HEK-mGAS clones. Loss of either p65 or β-catenin significantly reduced proliferation of HEK-mGAS clones, indicating that both factors are required for the proliferative effects of progastrin. Lengths of colon crypts and levels of p65, β-catenin, DCAMKL+1, and CD44 were significantly higher in ANXA2(+/+) mice compared with either ANXA2(-/-) mice given progastrin or ANXA2(+/+) and ANXA2(-/-) mice given saline. AnxA2 expression is required for the biologic effects of progastrin in vivo and in vitro and mediates the stimulatory effect of progastrin on p65NF-κ, β-catenin, and the putative stem cell markers DCAMKL+1 and CD44. AnxA2 might therefore mediate the hyperproliferative and cocarcinogenic effects of progastrin.

A Centrosomal Cdc20-APC Pathway Controls Dendrite Morphogenesis in Postmitotic Neurons

The ubiquitin ligase anaphase-promoting complex (APC) recruits the coactivator Cdc20 to drive mitosis in cycling cells. However, the nonmitotic functions of Cdc20-APC have remained unexplored. We report that Cdc20-APC plays an essential role in dendrite morphogenesis in postmitotic neurons. Knockdown of Cdc20 in cerebellar slices and in postnatal rats in vivo profoundly impairs the formation of granule neuron dendrite arbors in the cerebellar cortex. Remarkably, Cdc20 is enriched at the centrosome in neurons, and the centrosomal localization is critical for Cdc20-dependent dendrite development. We also find that the centrosome-associated protein histone deacetylase 6 (HDAC6) promotes the polyubiquitination of Cdc20, stimulates the activity of centrosomal Cdc20-APC, and drives the differentiation of dendrites. These findings define a postmitotic function for Cdc20-APC in the morphogenesis of dendrites in the mammalian brain. The identification of a centrosomal Cdc20-APC ubiquitin signaling pathway holds important implications for diverse biological processes, including neuronal connectivity and plasticity.

Werner Syndrome Protein, WRN, Protects Cells from DNA Damage Induced by the Benzene Metabolite Hydroquinone

Werner syndrome (WS) is a rare autosomal progeroid disorder caused by a mutation in the gene encoding the WRN (Werner syndrome protein), a member of the RecQ family of helicases with a role in maintaining genomic stability. Genetic association studies have previously suggested a link between WRN and susceptibility to benzene-induced hematotoxicity. To further explore the role of WRN in benzene-induced hematotoxicity, we used short hairpin RNA to silence endogenous levels of WRN in the human HL60 acute promyelocytic cell line and subsequently exposed the cells to hydroquinone (HQ). Suppression of WRN led to an accelerated cell growth rate, increased susceptibility to hydroquinone-induced cytotoxicity and genotoxicity as measured by the single-cell gel electrophoresis assay, and an enhanced DNA damage response. More specifically, loss of WRN resulted in higher levels of early apoptosis, marked by increases in relative levels of cleaved caspase-7 and cleaved poly (ADP-ribose) polymerase 1, in cells treated with HQ compared with control cells. Our data suggests that WRN plays an important role in the surveillance of and protection against DNA damage induced by HQ. This provides mechanistic support for the link between WRN and benzene-induced hematotoxicity.

The Presenilin 1 ΔE9 Mutation Gives Enhanced Basal Phospholipase C Activity and a Resultant Increase in Intracellular Calcium Concentrations

We studied effects of the familial Alzheimer's disease presenilin 1 (PS1) exon 9 deletion (PS1-DeltaE9) mutation on basal and carbachol-stimulated phosphoinositide (PI) hydrolysis and intracellular Ca(2+) concentrations ([Ca(2+)](i)) in human SH-SY5Y neuroblastoma cells. We demonstrate that PS1-DeltaE9 cells have an enhanced basal PI hydrolysis and [Ca(2+)](i) as compared with both wild type PS1 (PS1-WT) and nontransfected (NT) cells. Both were reversed by the phospholipase C (PLC) inhibitor neomycin. The PS1-DeltaE9-related high basal [Ca(2+)](i) was also reversed by xestospongin C confirming that this effect was inositol trisphosphate receptor-mediated. Carbachol gave a greater stimulation of [Ca(2+)](i) in PS1-DeltaE9 cells that took longer to return to basal as compared with responses seen in NT and PS1-WT cells. This long tail-off effect seen in PS1-DeltaE9 cells after carbachol stimulation was reversed by xestospongin C and dantrolene, suggesting that it was mediated by inositol trisphosphate receptor and ryanodine receptor amplification of Ca(2+). Ruthenium red only reduced carbachol peak elevations of [Ca(2+)](i) in NT and PS1-WT cells and not in PS1-DeltaE9 cells. No significant between cell type differences were seen for basal and carbachol-stimulated [Ca(2+)](i) with either ryanodine or the endoplasmic reticulum Ca(2+) ATPase inhibitor cyclopiazonic acid. Immunostaining experiments revealed that for all the cell types PS1 is present at the plasma membrane and co-localizes with N-cadherin, a component of the cell-cell adhesion complex. Immunoblotting of cell extracts for PLC-beta1 showed that, compared with NT and PS1-WT cells, the PS1-DeltaE9 transfectants gave a relative increase in levels of the calpain generated N-terminal fragment (100 kDa) over full-length (150 kDa) PLC-beta1. Our results suggest that the PS1-DeltaE9 mutation causes upstream changes in PI signaling with enhanced basal PLC activity as a primary effect that leads to a higher [Ca(2+)](i). This may provide a novel mechanism by which the PS1-DeltaE9 mutation sensitizes cells to apoptotic stimuli and enhanced amyloid beta generation.

Presence of Activated Antigen-Binding B Cells During Immunization Enhances Relative Levels of IFN-γ in T Cell Responses

AbstractTo examine the influence of Ag presentation by B cells on immune responses, we have used mice transgenic for an Ig heavy chain from a monoclonal anti-azobenzenearsonate (Ars) Ab to deliver Ag to B cells during immunization. A large proportion of transgene-expressing B cells in these mice binds Ars, while transgenic serum Ig shows poor Ars binding. Transgenic B cells present Ars proteins better than their nonhaptenated counterparts. This is associated with an increase in the proliferative responses of transgenic T cells to Ars protein immunization. Although B cell numbers in the transgenic mice are lower, many B cells in them show an activated phenotype, as identified by altered surface levels of peanut agglutinin reactivity, CD23, CD24, CD44, CD62L, and CD86. Even against nonhaptenated immunogens, transgenic responses show significant enhancement in the relative proportions of the Th1 cytokine IFN-γ over the Th2 cytokines IL-4 and IL-10. Haptenated immunogens further enhance the predilection of transgenic mice to produce relatively more IFN-γ. Consistent with this, there is an increase in IgG2a/IgG1 ratios in serum Abs in response to haptenated immunogens in transgenic mice. Adoptive transfer of primed hapten-specific secondary B cells into nontransgenic mice also induces an increase in relative levels of IFN-γ in response to haptenated immunogens. Thus, presentation of immunogen in vivo by activated Ag-binding B cells contributes to enhanced immunogenicity and a Th1 cytokine bias.

Expression of estrogen receptor-like immunoreactivity by different subgroups of basal forebrain cholinergic neurons in gonadectomized male and female rats

Recent studies have demonstrated that estrogen administration can produce significant increases in relative levels of choline acetyltransferase (ChAT) mRNA and protein in specific regions of the female, but not the male, rat basal forebrain. In the present study immunocytochemical techniques were used to identify and compare relative numbers of cholinergic neurons containing estrogen receptors within the medial septum, horizontal limb of the diagonal band of Broca, nucleus basalis magnocellularis, and striatum of gonadectomized male and female rats to determine whether there are differences in the percentage of cholinergic neurons expressing estrogen receptors which might contribute to the different regional- and sex-specific effects of estrogen which have been described. Counts of choline acetyltransferase-immunoreactive cells revealed significant regional differences in the average number of cholinergic neurons/section; however, no difference between males and females in the numbers of cholinergic neurons in each of the four regions analyzed was observed. Fifty to eighty percent of the cholinergic neurons detected in both males and females contained estrogen receptor-like immunoreactivity. A small but significant difference between males and females was detected with females having slightly more (10.5%) double-labeled cells than males overall. Individual comparisons revealed that significantly more (18–33%) double-labeled cells were detected in the horizontal limb of the diagonal band, but not in the medial septum, nucleus basalis, or striatum of females vs. males. There was also a small but significant regional difference in the percentage of double-labeled cells with the highest percentage (74.2%) detected in the striatum and the lowest percentage (63.4%) detected in the horizontal limb. None of these differences appear to account for the regional- and sex-specific effects of estrogen on cholinergic neurons which have been observed. We conclude that differences in the effects of estrogen on cholinergic neurons in males vs. females and in different Subregions of the female basal forebrain are not due to differences in the percentage of cholinergic neurons expressing estrogen receptors.