Multiplexed immunoassays for proteins using magnetic luminescent nanoparticles for internal calibration

Suspension arrays present a promising tool for multiplexed assays in large-scale screening applications. A simple and robust platform for quantitative multiprotein immunoanalysis has been developed with the use of magnetic Co:Nd:Fe(2)O(3)/luminescent Eu:Gd(2)O(3) core/shell nanoparticles (MLNPs) as a carrier. The magnetic properties of the MLNPs allow their manipulation by an external magnetic field in the separation and washing steps in the immunoassay. Their optical properties enable the internal calibration of the detection system. The multiplexed sandwich immunoassay involves dual binding events on the surface of the MLNPs functionalized with the capture antibodies. Secondary antibodies labeled with conventional organic dyes (Alexa Fluor) are used as reporters. The amount of the bound secondary antibody is directly proportional to the concentration of the analyte in the sample. In our approach, the fluorescence intensity of the reporter dye is related to the luminescence signal of the MLNPs. In this way, the intrinsic luminescence of the MLNPs serves as an internal standard in the quantitative immunoassay. The concept is demonstrated for a simultaneous immunoassay for three model proteins (human, rabbit, and mouse IgGs). The method uses a standard bench plate reader. It can be applied to disease diagnostics and to the detection of biological threats.     

A rapid assay for dihydropteroate synthase activity suitable for identification of inhibitors

The enzymes 6-hydroxymethylpterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) catalyze sequential steps in folate biosynthesis. They are present in microorganisms but absent in mammals and therefore are especially suitable targets for antimicrobials. Sulfa drugs (sulfonamides and sulfones) currently are used as antimicrobials targeting DHPS, although resistance to these drugs is increasing. The most widely used assay that measures activity of these enzymes, to assess new inhibitors in vitro, is not amenable to automation. This article describes a simple, coupled, enzymatic spectrophotometric assay where the product of the DHPS reaction, dihydropteroate, is reduced to tetrahydropteroate by excess dihydrofolate reductase (DHFR) using the cofactor NADPH. The oxidation of NADPH is monitored at 340 nm. The activity of both HPPK and DHPS can be measured in this assay, and it has been used to measure kinetic parameters of wild-type and sulfa drug-resistant DHPS enzymes to demonstrate the utility of the assay. It is a sensitive and reproducible assay that can be readily automated and used in multiwell plates. This NADPH-coupled microplate photometric assay could be used for rapid screening of chemical libraries for novel inhibitors of folate biosynthesis as the first step in developing new antimicrobial drugs targeting the folate biosynthetic pathway.     

Acute exercise stimulates macrophage function: possible role of NF-kappaB pathways

Moderate physical activity when performed on a regular basis presents a number of benefits to the whole organism, especially regarding immune system function, such as augmenting resistance to infections and to cancer growth. Although glutamine production by active muscle cells as well as neuroendocrine alterations mediated by the chronic adaptation to exercise may play a role, the entire mechanism by which exercise makes the immune system aware of challenges remains mostly uncovered. This is particularly true for the effects of an acute exercise session on immune function. In this work, circulating monocytes/macrophages from sedentary rats submitted to an acute (1 h) swimming session were tested for the ability of phagocytosing zymosan particles, phorbol myristate acetate (PMA)-induced hydrogen peroxide production, nitric oxide (NO) release (assessed by nitrate and nitrite production) and the expression of NO synthases (NOS-1, NOS-2 and NOS-3). The results showed that an exercise bout induced a 2.4-fold rise in macrophage phagocytic capacity (p = 0.0041), a 9.6-fold elevation in PMA-induced hydrogen peroxide release into the incubation media (1-h, p = 0.0022) and a 95.5%-augmentation in nitrite basal production (1-h incubation; p = 0.0220), which was associated with a marked expression of NOS-2 (the inducible NOS isoform; p = 0.0319), but not in other NOS gene products. Although NOS-2 expression is nuclear factor-kappaB (NF-kappaB)-dependent, no systemic oxidative stress was found, as inferred from the data of plasma TBARS and glutathione disulphide (GSSG) to glutathione (GSH) ratio in circulating blood erythrocytes which remained constant after the acute exercise. Also, no stressful situation seemed to be faced by monocytes/macrophages, since the expression of the 70-kDa heat shock protein (HSP70) remained unchanged. We conclude that NF-kappaB-dependent induction of NOS-2 and macrophage activation must be related to local factor(s) produced in the surroundings of monocytes/macrophages.     

QTL detection on porcine chromosome 12 for fatty-acid composition and association analyses of the fatty acid synthase, gastric inhibitory polypeptide and acetyl-coenzyme A carboxylase alpha genes

Refinement of previous QTL on porcine chromosome 12 for fatty-acid composition and a candidate gene association analysis were conducted using an Iberian × Landrace cross. The concentrations of ten fatty acids were assayed in backfat tissue from which four metabolic ratios were calculated for 403 F₂ animals. Linkage analysis identified two significant QTL. The first QTL was associated with the average chain length ratio and the percentages of myristic, palmitic and gadoleic acids. The second QTL was associated with percentages of palmitoleic, stearic and vaccenic acids. Based upon its position on SSC12, fatty acid synthase was tested as a candidate gene for the first QTL and no significant effects were found. Similarly, gastric inhibitory polypeptide ( GIP ) and acetyl-coenzyme A carboxylase alpha ( ACACA ) were tested as candidate genes for the second QTL using three SNPs in GIP and 15 synonymous SNPs in ACACA cDNA sequences. Two missense SNPs in GIP showed significant effects with palmitoleic and stearic fatty-acid concentration. Highly significant associations were found for two SNPs in ACACA with stearic, palmitoleic and vaccenic fatty-acid concentrations. These associations could be due to linkage disequilibrium with the causal mutations.     

Calmodulin Inhibitor-induced Apoptosis was Prevented by Glycogen Synthase Kinase-3 Inhibitors in PC12 Cells

Calmodulin is known to transduce Ca²⁺ signals by interacting with specific target proteins. In order to determine the role of calmodulin in regulating neuronal survival and death, we examined, whether calmodulin inhibitors induce caspase-dependent apoptotic cell death, and whether glycogen synthase kinase-3 is involved in calmodulin inhibitor-induced cell death in PC12 cells. W13, a calmodulin specific inhibitor increased apoptotic cell death with morphological changes characterized by cell shrinkage and nuclear condensation of fragmentation. Glycogen synthase kinase-3 inhibitors prevented calmodulin inhibitor-induced apoptosis. In addition, nerve growth factor and cycloheximide, a protein synthesis inhibitor, completely blocked cell death. Moreover, caspase-3 activation was accompanied by calmodulin inhibitor-induced cell death and inhibited by nerve growth factor. These results suggest that calmodulin inhibitors induce caspase-dependent apoptosis, and the activation of glycogen synthase kinase-3 is involved in the death of PC12 cells.     

An Inhibitory Role of Nitric Oxide in the Dynamic Regulation of the Blood-Brain Barrier Function

1. The present study aimed at elucidating the effect of nitric oxide (NO) on blood-brain barrier (BBB) function with mouse brain capillary endothelial (MBEC4) cells. 2. Histamine (20–100 μM) evoked NO production (1.6–7 μM) in MBEC4 cells in a dose-dependent manner. 3. The permeability coefficient of sodium fluorescein for MBEC4 cells and the cellular accumulation of rhodamine 123 in MBEC4 cells were increased dose-dependently by the addition of NO solutions (14 and 28 μM) every 10 min during a 30-min period. 4. The present study demonstrated that NO increased the permeability and inhibited the P-glycoprotein efflux pump of brain capillary endothelial cells, suggesting that NO plays an inhibitory role in the dynamic regulation of the BBB function.     

Inhibition of nitric oxide synthase enhances contractile response of ventricular myocytes from streptozotocin-diabetic rats

The contractile hyporesponsiveness of the streptozotocin diabetic rat heart in vitro to β-adrenergic agonists is eliminated when the heart is perfused with N^G-nitro-l-arginine methyl ester (l-NAME), a non-selective inhibitor of nitric oxide synthase (NOS). The following study evaluated the hypothesis that an increased production of NO/cGMP within the diabetic myocyte inhibits the β-adrenergic-stimulated increase in calcium current and contractile response. Male Sprague-Dawley rats were given an intravenous injection of streptozotocin (60 mg/kg). After 8 weeks, L-type calcium currents were recorded in ventricular myocytes using the whole cell voltage-clamp method. Shortening of isolated myocytes was determined using a video edge detection system. cAMP and cGMP were measured using radioimmunoassay. Nitric oxide production was determined using the Griess assay kit. Basal cGMP levels and nitric oxide production were elevated in diabetic myocytes. Shortening of the diabetic myocytes in response to isoproterenol (1 μM) was markedly diminished. However, there was no detectable difference in the isoproterenol-stimulated L-type calcium current or cAMP levels between control and diabetic myocytes. Acute superfusion of the diabetic myocyte with l-NAME (1 mM) decreased basal cGMP and markedly enhanced the shortening response to isoproterenol but did not alter isoproterenol-stimulated calcium current. These data suggest that increased production of NO/cGMP within the diabetic myocyte suppressed β-adrenergic stimulated shortening of the myocyte. However, NO/cGMP apparently does not suppress shortening of the myocyte by inhibition of the β-stimulated calcium current.     

Intestinal inflammation caused by magnesium deficiency alters basal and oxidative stress-induced intestinal function

The aim of this study was to determine the effect of magnesium deficiency on small intestinal morphology and function. Rats were assigned to 4 groups and placed on magnesium sufficient or deficient diet for 1 or 3 weeks. Infiltration of neutrophils and mucosal injury were assessed in stained sections of small intestine. Magnesium deficiency alone induced a significant increase in neutrophil infiltration and increased vascular ICAM-1 expression, in the absence of changes in mucosal injury or expression of proinflammatory mediators. Magnesium deficiency was associated with hyposecretory epithelial cell responses and vascular macromolecular leak in the small intestine and lung, which was attributed partly to reduced expression of NOS-3. To determine the effect of hypomagnesmia on the intestinal responses to a known oxidative stress, groups of rats were randomized to either sham operation or superior mesenteric artery occlusion for 10 (non-injurious) or 30 (injurious) minutes followed by a 1- or 4-hour reperfusion period. In response to mesenteric ischemia/reperfusion, deficient rats showed exaggerated PMN influx, but similar mucosal injury. Intestinal ischemia in sufficient animals induced vascular macromolecular leak in the small intestine and lung at 4 hours of reperfusion, with levels similar to those observed in untreated deficient rats. Acute magnesium repletion of deficient rats 24 h before surgery attenuated the exaggerated inflammation in deficient rats. These data show that magnesium deficiency induced a subclinical inflammation in the small intestine in the absence of mucosal injury, but with significant functional changes in local and remote organs and increased sensitivity to oxidative stress. The opinions contained herein are those of the authors and are not to be construed as official policy or reflecting the views of the Department of Defense     

Nitricoxide synthase-induced oxidative stress in prolonged alcoholic myopathies of rats

Previous studies showed that nitricoxide synthase (NOS) and oxidative stress can induce skeletal muscle atrophy in the muscular dystrophy and inclusion-body myopathy. There is a correlation between NOS and oxidative stress. However, it is not clear, whether there are some changes of the NOS activity in prolonged alcoholic myopathy (PAM), and whether NOS activity has relation to amyotrophy of PAM. We established experimental alcoholic myopathy model of rats by prolonged alcohol intake. We found that there is a reduction in GSH-px (P < 0.05) and an increase of SOD (P < 0.05), MDA (P < 0.05) and iNOS (P < 0.05) in the plantaris of the experimental group by spectrophotometer. In the soleus of the experimental group, except for MDA showed an increase (P < 0.05), the other enzymes showed no obvious difference (P > 0.05). The immunohistochemistry results showed that there was obvious expression of iNOS in the cytoplasm of plantaris in the experimental group and there was no expression of iNOS in the control group. There was a decrease of nNOS expression on the membranes of the plantaris cells in the experimental group by immunofluorescence. Meanwhile, we found the expression of nNOS in some cytoplasm. Our results suggested that NOS might be an important factor during the development of PAM. We could infer that there are some disturbances with regard to output and scavenging of free radical in PAM. Alcohol can induce the oxidative stress reaction and further result in imbalance of the oxidant-antioxidant status in the organism. Haiying Chu is the co-first author.     

The role of iNOS-derived NO in the antihypertrophic actions of B-type natriuretic peptide in neonatal rat cardiomyocytes

In the infarcted rat heart, the increase of NO occurs in the hypertrophied myocardium of non-infarcted areas and its antihypertrophic efficacy has been well established. As another endogenous regulator and the reliable index of heart pathology, B-type natriuretic peptide also exhibits the antihypertrophic properties in many tissues by elevating intracellular cGMP. Several studies indicate that natriuretic peptides family may exert some actions in part via a nitric oxide pathway following receptor-mediated stimulation of iNOS. Therefore, it raises our great interest to ask what role NO plays in the antihypertrophic actions of B-type natriuretic peptide in cardiomyocytes. Incubation of cardiomyocytes under mild hypoxia for 12 h caused a significant increase in cellular protein content, protein synthesis and cell surface sizes. This growth stimulation was suppressed by exogenous B-type natriuretic peptide in a concentration dependent manner. Furthermore, the generation of intracellular cGMP, the upregulation of iNOS mRNA expression, the increase of iNOS activity and subsequent nitrite generation in hypertrophic cardiomyocytes was also increased by B-type natriuretic peptide. AG, a selective iNOS inhibitor, inhibited the upregulation of iNOS expression and the increase of iNOS activity by the combination of B-type natriuretic peptide/mild hypoxia or by the combination of 8-bromo-cGMP/mild hypoxia. Rp-8-br-cGMP, cGMP dependent protein kinase inhibitor, attenuated the actions of B-type natriuretic peptide and 8-bromo-cGMP which increases intracellular cGMP independent of B-type natriuretic peptide. In conclusion, our present data suggest that B-type natriuretic peptide exerted the antihypertrophic effects in cardiomyocytes, which was partially attributed to induction of iNOS-derived NO by cGMP pathway.