Atherogenesis inhibition induced by magnesium-chloride fortification of drinking water

Magnesium (Mg) modulates blood lipid levels, atherogenesis, and atherosclerosis in rabbits, when supplemented to diet. We have recently reported that a high concentration (50 g/L) of Mg sulfate fortification of drinking water attenuates atherogenesis in male and female LDL-receptor-deficient mice fed a high-cholesterol diet. The aims of the current study were to examine whether lower concentrations and another Mg salt could also have such an antiatherogenic effect. Thirty male LDL-receptor-deficient mice were divided into three groups (n=10 in each group). The mice received either distilled water or water fortified with 0.83 g or with 8.3 g Mg-chloride per liter. In the first (27 wk) and second (5 wk) stages of the experiment, the mice received normal chow and Western-type diet, respectively. Blood was drawn for determination of plasma Mg, calcium, and lipid levels. The extent of atherosclerotic lesions was determined at the aortic sinus. Magnesium-chloride fortification of drinking water did not result in higher plasma Mg concentrations, whereas a trend toward lower plasma calcium concentrations did not reach statistical significance. Even though plasma lipid levels were similar at the beginning and the end of the study, there were decreased plasma cholesterol and triglyceride levels in the Mg groups after stage I. The atherosclerosis extent at the aortic sinus was significantly decreased in the 8.3-g Mg-chloride/L group (23,437 +/- 10,083 micron2) compared with the control group (65,937 +/- 31,761 microm2). There was also a trend toward lower atherosclerosis extent at the aortic sinus in the 0.83-g Mg-chloride/L group. An additional Mg salt (Mg-chloride) fortification of drinking water is capable of inhibiting atherogenesis in male LDL-receptor-deficient mice. That is done in a lower concentration of Mg than previously reported.     

Effect of hCG injection on prostaglandin E concentrations in ram seminal plasma

Ram and bull seminal plasma, respectively, contain 0.5-20 microg PGE/ml and 5-10 ng PGE/ml. To demonstrate that PGE concentrations in the seminal plasma are related to sperm quality and could be affected by hormonal stimulation in vivo, four rams were injected with 500 IU hCG, in and out of season. The rams responded 1 week after hCG with a 1.5- to 4-fold increase in seminal plasma PGE. The PGE peak was temporally separate from the hCG-induced rise in seminal plasma testosterone which was observed after 1 day. Using a simulated cryptochid ram, peaks in seminal fluid PGE were found to be associated with increased sperm velocity and sperm counts. In bulls, PGE concentrations in the seminal plasma of good bulls were significantly higher than that found in poor and cryptorchid bulls.     

A template-proximal RNA paired element contributes to Saccharomyces cerevisiae telomerase activity

The ribonucleoprotein complex telomerase is critical for replenishing chromosome-end sequence during eukaryotic DNA replication. The template for the addition of telomeric repeats is provided by the RNA component of telomerase. However, in budding yeast, little is known about the structure and function of most of the remainder of the telomerase RNA. Here, we report the identification of a paired element located immediately 5' of the template region in the Saccharomyces cerevisiae telomerase RNA. Mutations disrupting or replacing the helical element showed that this structure, but not its exact nucleotide sequence, is important for telomerase function in vivo and in vitro. Biochemical characterization of a paired element mutant showed that the mutant generated longer products and incorporated noncognate nucleotides. Sequencing of in vivo synthesized telomeres from this mutant showed that DNA synthesis proceeded beyond the normal template. Thus, the S. cerevisiae element resembles a similar element found in Kluyveromyces budding yeasts with respect to a function in template boundary specification. In addition, the in vitro activity of the paired element mutant indicates that the RNA element has additional functions in enzyme processivity and in directing template usage by telomerase.     

Folate deprivation results in the loss of breast cancer resistance protein (BCRP/ABCG2) expression. A role for BCRP in cellular folate homeostasis

Breast cancer resistance protein (BCRP/ABCG2) is currently the only ABC transporter that exports mono- and polyglutamates of folates and methotrexate (MTX). Here we explored the relationship between cellular folate status and BCRP expression. Toward this end, MCF-7 breast cancer cells, with low BCRP and moderate multidrug resistance protein 1 (MRP1/ABCC1) levels, and their mitoxantrone (MR)-resistant MCF-7/MR subline, with BCRP overexpression and low MRP1 levels, were gradually deprived of folic acid from 2.3 microm to 3 nm resulting in the sublines MCF-7/LF and MCF-7/MR-LF. These cell lines expressed only residual BCRP mRNA and protein levels and retained a poor MRP2 (ABCC2) through MRP5 (ABCC5) expression. Furthermore, MCF-7/MR-LF cells also displayed 5-fold decreased MRP1 levels relative to MCF-7/MR cells. In contrast, BCRP overexpression was largely retained in MCF-7/MR cells grown in MR-free medium containing 2.3 microm folic acid. Loss of BCRP expression in MCF-7/LF and MCF-7/MR-LF cells resulted in the following: (a) a prominent decrease in the efflux of Hoechst 33342, a BCRP substrate; (b) an approximately 2-fold increase in MR accumulation as revealed by flow cytometry; this was accompanied by a 2.5- and approximately 84-fold increased MR sensitivity in these cell lines, respectively. Consistently, Ko143, a specific BCRP inhibitor, rendered MCF-7 and MCF-7/MR cells 2.1- and approximately 16.4-fold more sensitive to MR, respectively. Loss of BCRP expression also resulted in the following: (c) an identical MTX sensitivity in these cell lines thereby losing the approximately 28-fold MTX resistance of the MCF-7/MR cells; (d) an approximately 2-fold increase in the 4- and 24-h accumulation of [(3)H]folic acid. Furthermore, MCF-7/MR-LF cells displayed a significant increase in folylpoly-gamma-glutamate synthetase activity. Hence, consistent with the mono- and polyglutamate folate exporter function of BCRP, down-regulation of BCRP and increased folylpoly-gamma-glutamate synthetase activity appear to be crucial components of cellular adaptation to folate deficiency conditions. This is the first evidence for the possible role of BCRP in the maintenance of cellular folate homeostasis.     

Monitoring the apoptotic process induced by oxidized low-density lipoprotein in Jurkat T-lymphoblast and U937 monocytic human cell lines

Cell death is a major event in the pathophysiology of atherosclerosis. Oxidized low-density lipoprotein (Ox-LDL), which plays a key role in the atherogenesis, has a powerful cytotoxic effect and causes necrosis or apoptosis of different types of cells. In the present work we studied the mechanism of cell death in two model systems: T lymphocytes and monocytes cell line, exposed to Ox-LDL. Ox-LDL, but not native low-density lipoprotein (LDL), was found to be cytotoxic to both cell types in a dose and time dependent manner. Apoptotic cell deat was analyzed by evaluating cell size, nucleus DNA content and plasma membrane asymmetry. Early cytoplasmic condensation resulting from cell shrinkage was measured by monitoring fluorescence polarization (FP) of fluorescein labeled cells. The redical scavenger superoxide dismutase (SOD), in a time- and dose-dependent manner, reduced the apoptotic effect of Ox-LDL. Hyperpolarization of fluorescein-labeled cells preceded the appearance of phosphatidylserine (PS) on the plasma membrane. This sensitive parameter for early apoptosis detected different cell death kinetics, as well as varying sensitivity to the inhibitory effect of SOD in monocytes and lymphocytes. Such data suggest that reactive oxygen species generation are involved, in Ox-LDL-induced apoptosis and that monocytes are more susceptible to cell death triggered by oxidative stress.     

Comparative sequence analysis and patterns of covariation in RNA secondary structures

A novel method of RNA secondary structure prediction based on a comparison of nucleotide sequences is described. This method correctly predicts nearly all evolutionarily conserved secondary structures of five different RNAs: tRNA, 5S rRNA, bacterial ribonuclease P (RNase P) RNA, eukaryotic small subunit rRNA, and the 3' untranslated region (UTR) of the Drosophila bicoid (bcd) mRNA. Furthermore, covariations occurring in the helices of these conserved RNA structures are analyzed. Two physical parameters are found to be important determinants of the evolution of compensatory mutations: the length of a helix and the distance between base-pairing nucleotides. For the helices of bcd 3' UTR mRNA and RNase P RNA, a positive correlation between the rate of compensatory evolution and helix length is found. The analysis of Drosophila bcd 3' UTR mRNA further revealed that the rate of compensatory evolution decreases with the physical distance between base-pairing residues. This result is in qualitative agreement with Kimura's model of compensatory fitness interactions, which assumes that mutations occurring in RNA helices are individually deleterious but become neutral in appropriate combinations.     

ppk23-Dependent chemosensory functions contribute to courtship behavior in Drosophila melanogaster

Insects utilize diverse families of ion channels to respond to environmental cues and control mating, feeding, and the response to threats. Although degenerin/epithelial sodium channels (DEG/ENaC) represent one of the largest families of ion channels in Drosophila melanogaster, the physiological functions of these proteins are still poorly understood. We found that the DEG/ENaC channel ppk23 is expressed in a subpopulation of sexually dimorphic gustatory-like chemosensory bristles that are distinct from those expressing feeding-related gustatory receptors. Disrupting ppk23 or inhibiting activity of ppk23-expressing neurons did not alter gustatory responses. Instead, blocking ppk23-positive neurons or mutating the ppk23 gene delayed the initiation and reduced the intensity of male courtship. Furthermore, mutations in ppk23 altered the behavioral response of males to the female-specific aphrodisiac pheromone 7(Z), 11(Z)-Heptacosadiene. Together, these data indicate that ppk23 and the cells expressing it play an important role in the peripheral sensory system that determines sexual behavior in Drosophila.     

Measuring adiposity in patients: the utility of body mass index (BMI), percent body fat, and leptin

Background

Obesity is a serious disease that is associated with an increased risk of diabetes, hypertension, heart disease, stroke, and cancer, among other diseases. The United States Centers for Disease Control and Prevention (CDC) estimates a 20% obesity rate in the 50 states, with 12 states having rates of over 30%. Currently, the body mass index (BMI) is most commonly used to determine adiposity. However, BMI presents as an inaccurate obesity classification method that underestimates the epidemic and contributes to failed treatment. In this study, we examine the effectiveness of precise biomarkers and duel-energy x-ray absorptiometry (DXA) to help diagnose and treat obesity.

Methodology/Principal Findings

A cross-sectional study of adults with BMI, DXA, fasting leptin and insulin results were measured from 1998–2009. Of the participants, 63% were females, 37% were males, 75% white, with a mean age = 51.4 (SD = 14.2). Mean BMI was 27.3 (SD = 5.9) and mean percent body fat was 31.3% (SD = 9.3). BMI characterized 26% of the subjects as obese, while DXA indicated that 64% of them were obese. 39% of the subjects were classified as non-obese by BMI, but were found to be obese by DXA. BMI misclassified 25% men and 48% women. Meanwhile, a strong relationship was demonstrated between increased leptin and increased body fat.

Conclusions/Significance

Our results demonstrate the prevalence of false-negative BMIs, increased misclassifications in women of advancing age, and the reliability of gender-specific revised BMI cutoffs. BMI underestimates obesity prevalence, especially in women with high leptin levels (>30 ng/mL). Clinicians can use leptin-revised levels to enhance the accuracy of BMI estimates of percentage body fat when DXA is unavailable.     

Quantitative analysis of caveolin-rich lipid raft proteins from primary and metastatic colorectal cancer clones

Caveolin-rich lipid rafts (CLRs) are thickened sections of the cell membrane that are composed of the integral membrane proteins caveolins together with saturated long chain fatty acids, cholesterol and lipids. Membrane proteins - lipid raft proteins in particular - may play important roles in cell signaling and cell-cell interaction. Due to their unique structure, CLRs seem to be the preferred docking site for specific proteins involved in focal adhesion and cancer metastasis. Our objective was thus to identify and quantify CLR proteins from primary and metastatic colorectal cancer (CRC) clones. We found differential expression of nine CLR proteins from primary and metastatic CRC clones. Among the identified proteins, an immune system inhibiting protein was significantly overexpressed in the metastatic clone, while cell adhesion and transport molecules were among the overexpressed proteins in the primary clone. All the identified CRL proteins are involved in tumorigenesis, specifically metastasis, and may thus serve as therapeutic targets. A novel concept for identification and quantification of CLR proteins with label-free mass spectrometry method was specifically examined in this study. Validation of the method against immunoblotting and FACS analysis indicates that it can be applied for the identification of novel biomarkers for cancer and metastasis.