Down-regulation of human topoisomerase IIα expression correlates with relative amounts of specificity factors Sp1 and Sp3 bound at proximal and distal promoter regions

<p>Abstract</p> <p>Background</p> <p>Topoisomerase IIα has been shown to be down-regulated in doxorubicin-resistant cell lines. The specificity proteins Sp1 and Sp3 have been implicated in regulation of topoisomerase IIα transcription, although the mechanism by which they regulate expression is not fully understood. Sp1 has been shown to bind specifically to both proximal and distal GC elements of the human topoisomerase IIα promoter <it>in vitro</it>, while Sp3 binds only to the distal GC element unless additional flanking sequences are included. While Sp1 is thought to be an activator of human topoisomerase IIα, the functional significance of Sp3 binding is not known. Therefore, we sought to determine the functional relationship between Sp1 and Sp3 binding to the topoisomerase IIα promoter <it>in vivo</it>. We investigated endogenous levels of Sp1, Sp3 and topoisomerase IIα as well as binding of both Sp1 and Sp3 to the GC boxes of the topoisomerase IIα promoter in breast cancer cell lines <it>in vivo </it>after short term doxorubicin exposure.</p> <p>Results</p> <p>Functional effects of Sp1 and Sp3 were studied using transient cotransfection assays using a topoisomerase IIα promoter reporter construct. The <it>in vivo </it>interactions of Sp1 and Sp3 with the GC elements of the topoisomerase IIα promoter were studied in doxorubicin-treated breast cancer cell lines using chromatin immunoprecipitation assays. Relative amounts of endogenous proteins were measured using immunoblotting. <it>In vivo </it>DNA looping mediated by proteins bound at the GC1 and GC2 elements was studied using the chromatin conformation capture assay. Both Sp1 and Sp3 bound to the GC1 and GC2 regions. Sp1 and Sp3 were transcriptional activators and repressors respectively, with Sp3 repression being dominant over Sp1-mediated activation. The GC1 and GC2 elements are linked <it>in vivo </it>to form a loop, thus bringing distal regulatory elements and their cognate transcription factors into close proximity with the transcription start site.</p> <p>Conclusion</p> <p>These observations provide a mechanistic explanation for the modulation of topoisomerase IIα and concomitant down-regulation that can be mediated by topoisomerase II poisons. Competition between Sp1 and Sp3 for the same cognate DNA would result in activation or repression depending on absolute amounts of each transcription factor in cells treated with doxorubicin.</p

Structural characterisation of neutrophil glycans by ultra sensitive mass spectrometric glycomics methodology

Neutrophils are the most abundant white blood cells in humans and play a vital role in several aspects of the immune response. Numerous reports have implicated neutrophil glycosylation as an important factor in mediating these interactions. We report here the application of high sensitivity glycomics methodologies, including matrix assisted laser desorption ionisation (MALDI-TOF) and MALDI-TOF/TOF analyses, to the structural analysis of N- and O-linked carbohydrates released from two samples of neutrophils, prepared by two separate and geographically remote laboratories. The data produced demonstrates that the cells display a diverse range of sialylated and fucosylated complex glycans, with a high level of similarity between the two preparations

Allele-specific differences in ryanodine receptor 1 mRNA expression levels may contribute to phenotypic variability in malignant hyperthermia

<p>Abstract</p> <p>Background</p> <p>Malignant hyperthermia (MH) is a dominantly inherited skeletal muscle disorder that can cause a fatal hypermetabolic reaction to general anaesthetics. The primary locus of MH (MHS1 locus) in humans is linked to chromosome 19q13.1, the position of the gene encoding the ryanodine receptor skeletal muscle calcium release channel (RyR1).</p> <p>Methods</p> <p>In this study, an inexpensive allele-specific PCR (AS-PCR) assay was designed that allowed the relative quantification of the two RyR1 transcripts in heterozygous samples found to be susceptible to MH (MHS). Allele-specific differences in RyR1 expression levels can provide insight into the observed variable penetrance and variations in MH phenotypes between individuals. The presence/absence of the H4833Y mutation in <it>RYR</it>1 transcripts was employed as a marker that allowed discrimination between the two alleles.</p> <p>Results</p> <p>In four skeletal muscle samples and two lymphoblastoid cell lines (LCLs) from different MHS patients, the wild type allele was found to be expressed at higher levels than the mutant RyR1 allele. For both LCLs, the ratios between the wild type and mutant <it>RYR</it>1 alleles did not change after different incubation times with actinomycin D. This suggests that there are no allele-specific differences in RyR1 mRNA stability, at least in these cells.</p> <p>Conclusion</p> <p>The data presented here revealed for the first time allele-specific differences in <it>RYR</it>1 mRNA expression levels in heterozygous MHS samples, and can at least in part contribute to the observed variable penetrance and variations in MH clinical phenotypes.</p

Malignant hyperthermia

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle that presents as a hypermetabolic response to potent volatile anesthetic gases such as halothane, sevoflurane, desflurane and the depolarizing muscle relaxant succinylcholine, and rarely, in humans, to stresses such as vigorous exercise and heat. The incidence of MH reactions ranges from 1:5,000 to 1:50,000–100,000 anesthesias. However, the prevalence of the genetic abnormalities may be as great as one in 3,000 individuals. MH affects humans, certain pig breeds, dogs, horses, and probably other animals. The classic signs of MH include hyperthermia to marked degree, tachycardia, tachypnea, increased carbon dioxide production, increased oxygen consumption, acidosis, muscle rigidity, and rhabdomyolysis, all related to a hypermetabolic response. The syndrome is likely to be fatal if untreated. Early recognition of the signs of MH, specifically elevation of end-expired carbon dioxide, provides the clinical diagnostic clues. In humans the syndrome is inherited in autosomal dominant pattern, while in pigs in autosomal recessive. The pathophysiologic changes of MH are due to uncontrolled rise of myoplasmic calcium, which activates biochemical processes related to muscle activation. Due to ATP depletion, the muscle membrane integrity is compromised leading to hyperkalemia and rhabdomyolysis. In most cases, the syndrome is caused by a defect in the ryanodine receptor. Over 90 mutations have been identified in the RYR-1 gene located on chromosome 19q13.1, and at least 25 are causal for MH. Diagnostic testing relies on assessing the in vitro contracture response of biopsied muscle to halothane, caffeine, and other drugs. Elucidation of the genetic changes has led to the introduction, on a limited basis so far, of genetic testing for susceptibility to MH. As the sensitivity of genetic testing increases, molecular genetics will be used for identifying those at risk with greater frequency. Dantrolene sodium is a specific antagonist of the pathophysiologic changes of MH and should be available wherever general anesthesia is administered. Thanks to the dramatic progress in understanding the clinical manifestation and pathophysiology of the syndrome, the mortality from MH has dropped from over 80% thirty years ago to less than 5%

Effect of a Combined Walking and Conversation Intervention on Functional Mobility of Nursing Home Residents With Alzheimer Disease

Assisted walking and walking combined with conversation were compared to a conversation-only intervention in nursing home residents with Alzheimer disease. Sixty-five subjects randomly assigned to treatment group were tested at baseline and end of treatment. Subjects mean Mini-Mental State Examination score was 10.83; mean age was 87. Treatment was given for 30 minutes three times a week for 16 weeks. Subjects in the assisted walking group declined 20.9% in functional mobility; the conversation group declined 18.8%. The combined walking and conversation treatment group declined only 2.5%. These differences in outcome were significant and appear to have been affected by differences in treatment fidelity. Subjects in the conversation treatment group completed 90% of intended treatment compared with 75% in the combined group and only 57% in the assisted walking group. Failure to treat was due to subject refusal and physical illness. The conversation component of the combined walking and conversation treatment intervention appears to have improved compliance with the intervention, thereby improving treatment outcome. Results indicate that assisted walking with conversation can contribute to maintenance of functional mobility in institutionalized populations with Alzheimer disease. Staff assigned to this task should be prepared to use effective communication strategies to gain acceptance of the intervention

The proximal topoisomerase IIα promoter forms a loop between GC1 and GC2

<p><b>Copyright information:</b></p><p>Taken from "Down-regulation of human topoisomerase IIα expression correlates with relative amounts of specificity factors Sp1 and Sp3 bound at proximal and distal promoter regions"</p><p>http://www.biomedcentral.com/1471-2199/8/36</p><p>BMC Molecular Biology 2007;8():36-36.</p><p>Published online 20 May 2007</p><p>PMCID:PMC1885802.</p><p></p> A ligation product (429 bp) was detected by PCR (lane 2) after completion of the 3C assay. Digestion of ligated product (lane 1) with 36 I yielded two fragments (123 bp and 306 bp). The PCR product from the uncut genomic DNA control (lane 4) was 571 bp in size. When digested with 36 I, genomic DNA product (lane 3) yielded three fragments (123, 306 and 142 bp) as predicted from the DNA sequence

Transcriptional modulation of topoisomerase IIα facilitated by DNA looping

<p><b>Copyright information:</b></p><p>Taken from "Down-regulation of human topoisomerase IIα expression correlates with relative amounts of specificity factors Sp1 and Sp3 bound at proximal and distal promoter regions"</p><p>http://www.biomedcentral.com/1471-2199/8/36</p><p>BMC Molecular Biology 2007;8():36-36.</p><p>Published online 20 May 2007</p><p>PMCID:PMC1885802.</p><p></p> (A) High levels of Sp1 and the formation of Sp1 multimers results in increased transcriptional activity of the topoisomerase IIα promoter. (B) A repressive effect on promoter activity when Sp1/3 levels are equal as Sp1 can no longer form protein/protein interactions because of Sp3 occupancy at either GC1 or GC2. (C) Higher Sp3 levels show a dominant repressive effect of promoter activity as most GC1 and GC2 elements are occupied by Sp3

Initiation and Regulation of Complement during Hemolytic Transfusion Reactions

Hemolytic transfusion reactions represent one of the most common causes of transfusion-related mortality. Although many factors influence hemolytic transfusion reactions, complement activation represents one of the most common features associated with fatality. In this paper we will focus on the role of complement in initiating and regulating hemolytic transfusion reactions and will discuss potential strategies aimed at mitigating or favorably modulating complement during incompatible red blood cell transfusions

Lung defense through interleukin-8 carries a cost of chronic lung remodeling and impaired function

RATIONALE: IL-8 dependent inflammation is a hallmark of host lung innate immunity to bacterial pathogens, yet in many human lung diseases including COPD, bronchiectasis, and pulmonary fibrosis, there are progressive, irreversible pathologic, changes associated with elevated levels of IL-8 in the lung. OBJECTIVES: To better understand the duality of IL-8 dependent host immunity to bacterial infection and lung pathology, we targeted human IL-8 to express transgenically in murine bronchial epithelium, investigating the impact of over-expression on lung bacterial clearance, host immunity, lung pathology and function. MEASUREMENTS AND MAIN RESULTS: Persistent IL-8 expression in bronchial epithelium resulted in neutrophilia, neutrophil maturation, activation and chemtoaxis. There was enhanced protection from challenge with Pseudomonas aeruginosa and significant changes in baseline expression of innate and adaptive immunity transcripts for Ccl5, Tlr6, IL2 and Tlr1. There was increased expression of Tbet and Foxp3 in response to the Pseudomonas antigen, OprF, indicating a regulatory T cell phenotype. However, this enhanced bacterial immunity comes at the high price of progressive lung remodelling, with increased inflammation, mucus hyper-secretion, and fibrosis. There is increased expression of Ccl3 and reduced expressioh of Claudin 18 and F11r, with damage to epithelial organization leading to leaky tight junctions, all resulting in impaired lung function with reduced compliance, increased resistance and bronchial hyperreactivity measured by whole body plethysmography. CONCLUSIONS: IL-8 over-expression in the bronchial epithelium benefits lung immunity to bacterial infection, but specifically drives lung damage through persistent inflammation, lung remodelling and damaged tight junctions, leading to impaired lung function