Monitoring gene expression: quantitative real-time rt-PCR.

Two-step quantitative real-time RT-PCR (RT-qPCR), also known as real-time RT-PCR, kinetic RT-PCR, or quantitative fluorescent RT-PCR, has become the method of choice for gene expression analysis during the last few years. It is a fast and convenient PCR method that combines traditional RT-PCR with the phenomenon of fluorescence resonance energy transfer (FRET) using fluorogenic primers. The detection of changes in fluorescence intensity during the reaction enables the user to follow the PCR reaction in real time.

Dietary alpha-cyclodextrin lowers low-density lipoprotein cholesterol and alters plasma fatty acid profile in low-density lipoprotein receptor knockout mice on a high-fat diet.

High dietary intake of saturated fat and cholesterol, and elevated low-density lipoprotein cholesterol levels are some of the modifiable risk factors for cardiovascular disease. Alpha-cyclodextrin (a-CD) when given orally has been shown in rats to increase fecal saturated fat excretion and to reduce blood total cholesterol levels in obese hypertriglyceridemic subjects with type 2 diabetes mellitus. In this study, the effects of dietary a-CD on lipid metabolism in low-density lipoprotein receptor knockout mice were investigated.

Central quadrantectomy with resection of the nipple-areola complex compared with mastectomy in patients with retroareolar breast cancer.

Appropriate surgery in women with retroareolar breast cancer should allow resection of the cancer with wide free margins and an acceptable cosmetic result. The aim of this study was to compare breast conservation surgery (BCS) to mastectomy for treatment of retroareolar breast cancer. In a prospective nonrandomized study, 69 women with retroareolar breast cancers underwent either central quadrantectomy (n=33) with complete removal of the nipple-areola complex or mastectomy (n=36).

Oncoplastic techniques allow breast-conserving treatment in centrally located breast cancers.

Background: Operative techniques for oncoplastic reconstruction combine oncologic extirpation of the tumor with immediate reconstruction of breast shape and symmetry. These techniques are increasingly being used for breast-conservation therapy of centrally located breast carcinomas. The goal of this study was to provide an overview of the various surgical options for oncoplastic treatment of central breast carcinomas.

Enhanced ABCG1 expression increases atherosclerosis in LDLr-KO mice on a western diet.

ABCG1 promotes cholesterol efflux from cells, but ABCG1(-/-) bone marrow transplant into ApoE(-/-) and LDLr(-/-) mice reduces atherosclerosis. To further investigate the role of ABCG1 in atherosclerosis, ABCG1 transgenic mice were crossed with LDLr-KO mice and placed on a high-fat western diet. Increased expression of ABCG1 mRNA was detected in liver (1.

ABCA1 overexpression in the liver of LDLr-KO mice leads to accumulation of pro-atherogenic lipoproteins and enhanced atherosclerosis.

The identification of ABCA1 as a key transporter responsible for cellular lipid efflux has led to considerable interest in defining its role in cholesterol metabolism and atherosclerosis. In this study, the effect of overexpressing ABCA1 in the liver of LDLr-KO mice was investigated. Compared with LDLr-KO mice, ABCA1-Tg x LDLr-KO (ABCA1-Tg) mice had significantly increased plasma cholesterol levels, mostly because of a 2.

Compound heterozygosity (G71R/R140H) in the lecithin:cholesterol acyltransferase (LCAT) gene results in an intermediate phenotype between LCAT-deficiency and fish-eye disease.

The esterification of free cholesterol (FC) in plasma, catalyzed by the enzyme lecithin:cholesterol acyltransferase (LCAT; EC 2.3.1.

Endothelial lipase provides an alternative pathway for FFA uptake in lipoprotein lipase-deficient mouse adipose tissue.

Lipoprotein lipase (LPL) is thought to be the only enzyme responsible for the catabolism of triglycerides (TGs) associated with TG-rich lipoproteins in adipose tissue (AT). However, LPL deficiency in humans and induced mutant mice is not associated with decreased fat mass. We investigated whether endothelial lipase (EL), a recently discovered phospholipase, might represent an alternative mechanism for the uptake of phospholipid-derived fatty acids in murine lipoprotein-deficient AT.

Hepatic lipase, lipoprotein metabolism, and atherogenesis.

The role of hepatic lipase as a multifunctional protein that modulates lipoprotein metabolism and atherosclerosis has been extensively documented over the last decade. Hepatic lipase functions as a lipolytic enzyme that hydrolyzes triglycerides and phospholipids present in circulating plasma lipoproteins. Hepatic lipase also serves as a ligand that facilitates lipoprotein uptake by cell surface receptors and proteoglycans, thereby directly affecting cellular lipid delivery.

Dependence of lipoprotein-lipase-catalyzed triacylglycerol hydrolysis on droplet size of synthetic monodisperse emulsions measured with static light scattering.

Well-defined triolein emulsions of low polydispersity were prepared by shearing a crude emulsion in a modified Couette cell, resulting in radii in the range of 300 to 900 nm. These emulsions were used as synthetic substrates for lipoprotein lipase, a key enzyme for the hydrolysis of serum triacylglycerols. The change in radius with time was studied with on-line static light scattering at 37 degrees C.

Dopamine receptor D3 mRNA expression in human lymphocytes is negatively correlated with the personality trait of persistence.

It has been proposed that neurotransmitter receptor expression in peripheral immune cells reflects expression of these receptors in the brain. To test this "peripheral marker hypothesis", we compared mRNA expression of the dopamine receptors D3 (DRD3) and D4 (DRD4) in peripheral blood lymphocytes (PBL) to personality traits assessed with the Temperament and Character Inventory (TCI) in 50 healthy and unmedicated Caucasian individuals. A shared variance of at least 17% (p=0.

Reduced dopamine D3 receptor expression in blood lymphocytes of smokers is negatively correlated with daily number of smoked cigarettes: a peripheral correlate of dopaminergic alterations in smokers.

The mesolimbic dopaminergic system is known to mediate rewarding effects of nicotine administration, and dysfunctions of this system may underlie failure to stop cigarette smoking. Expression of dopamine receptors in peripheral blood lymphocytes (PBLs) has been indicated as a peripheral correlate of brain status. Dopamine receptor D(3) (DRD3) and D(4) (DRD4) mRNA expression in PBLs was measured by real-time polymerase chain reaction in smokers (n=26) and former smokers (n=14), compared with nonsmoking control subjects (n=35).

Reduced dopamine D4 receptor mRNA expression in lymphocytes of long-term abstinent alcohol and heroin addicts.

Aim: It has been repeatedly suggested that dopamine receptor expression in peripheral blood lymphocytes reflects, to some extent, brain status. The aim of the present study was to investigate dopamine receptor expression in peripheral blood lymphocytes of long-term abstinent alcohol and heroin addicts against the background of the hypothesis, that a persisting dysfunction of the dopaminergic system contributes a biological cause to the chronic character of addiction.

Design: Dopamine D3 and D4 receptor mRNA expression in peripheral blood lymphocytes was measured by real-time polymerase chain reaction (PCR) in 19 alcohol addicts, abstinent for 6.

Defective uptake of triglyceride-associated fatty acids in adipose tissue causes the SREBP-1c-mediated induction of lipogenesis.

Lipoprotein lipase (LPL) is the only known enzyme in the capillary endothelium of peripheral tissues that hydrolizes plasma triglycerides and provides fatty acids (FAs) for their subsequent tissue uptake. Previously, we demonstrated that mice that express LPL exclusively in muscle develop essentially normal fat mass despite the absence of LPL and the deprivation of nutritionally derived FAs in adipose tissue (AT). Using this mouse model, we now investigated the metabolic response to LPL deficiency in AT that enables maintenance of normal AT mass.

Decreased fatty acid esterification compensates for the reduced lipolytic activity in hormone-sensitive lipase-deficient white adipose tissue.

It has been observed previously that hormone-sensitive lipase-deficient (HSL-ko) mice have reduced white adipose tissue (WAT) stores compared to control mice. These findings contradict the expectation that the decreased lipolytic activity in WAT of HSL-ko mice would cause accumulation of triglycerides (TGs) in that tissue. Here we demonstrate that the cellular TG synthesis in HSL-deficient WAT is markedly reduced due to downregulation of the enzymatic activities of glycerophosphate acyltransferase, dihydroxyacetonphosphate acyltransferase, lysophosphatidate acyltransferase, and diacylglycerol acyltransferase.

Transgenic mice expressing lipoprotein lipase in adipose tissue. Absence of the proximal 3'-untranslated region causes translational upregulation.

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein and adipocyte metabolism. Defects in LPL can lead to hypertriglyceridemia and the subsequent development of atherosclerosis. The mechanisms of regulation of this enzyme are complex and may occur at multiple levels of gene expression.

Effects of lipoprotein lipase on uptake and transcytosis of low density lipoprotein (LDL) and LDL-associated alpha-tocopherol in a porcine in vitro blood-brain barrier model.

During the present study the contribution of lipoprotein lipase (LPL) to low density lipoprotein (LDL) holoparticle and LDL-lipid (alpha-tocopherol (alphaTocH)) turnover in primary porcine brain capillary endothelial cells (BCECs) was investigated. The addition of increasing LPL concentrations to BCECs resulted in up to 11-fold higher LDL holoparticle cell association. LPL contributed to LDL holoparticle turnover, an effect that was substantially increased in response to LDL-receptor up-regulation.

Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis.

Hormone-sensitive lipase (HSL) is expressed predominantly in white and brown adipose tissue where it is believed to play a crucial role in the lipolysis of stored triglycerides (TG), thereby providing the body with energy substrate in the form of free fatty acids (FFA). From in vitro assays, HSL is known to hydrolyze TG, diglycerides (DG), cholesteryl esters, and retinyl esters. In the current study we have generated HSL knock-out mice and demonstrate three lines of evidence that HSL is instrumental in the catabolism of DG in vivo.

Formation of virus-like particles from cloned cDNAs of Thogoto virus.

Thogoto virus (THOV) is the type species of tick-transmitted orthomyxoviruses. Here, we describe the generation of virus-like particles (VLP) of THOV from cloned cDNAs. To synthesize the six structural proteins of THOV in mammalian cells, we used T7-controlled expression plasmids and a recombinant vaccinia virus producing T7 RNA polymerase.

Physical and genetic map of the genome of Staphylococcus carnosus TM300.

A genome map of Staphylococcus carnosus TM300, an important micro-organism in the food industry and long used as a starter culture, was constructed by pulsed-field gel electrophoresis of DNA fragments obtained after digestion with NotI, SfiI and ApaI. The size of the chromosome was estimated to be 2590 kb. The fragments were assembled into a physical map using a combination of complementary methods including multiple and partial digests of genomic DNA, hybridization with homologous gene probes, and cross-Southern hybridization.