Immunoassay quantification using surface-enhanced fluorescence (SEF) tags.

Fluorescence-based immonoassays are widely used in several areas, ranging from basic biomedical research to disease diagnostics. A variety of new probes have been developed recently to address some limitations in typical assays performed with organic dyes. Ideally, new fluorescence tags that allow quantification with a low limit of detection are highly desired. In this work, the surface-enhanced fluorescence (SEF) phenomenon was explored in the development of tags for Immunoglobulin-M (IgM) detection. Shell-isolated gold nanoparticles (Au-SHINs) with 100 nm core size and a 10 nm silica shell were synthesized. These particles contain an outermost thin fluorescent layer of nile blue (NB) that was further coated by another 5 nm of silica (SEF tags). The outer silica shell was then functionalized with antibodies to allow the detection of IgM as in typical immunological sandwich assays. IgM concentrations down to the 10 ng mL-1 mark were successfully detected. A linear dependence between the average fluorescence intensity and the IgM concentration was found.

Unveiling the mechanisms underlying photothermal efficiency of gold shell-isolated nanoparticles (AuSHINs) on ductal mammary carcinoma cells (BT-474).

Gold nanoparticles are valuable photothermal agents owing to their efficient photothermal conversion, photobleaching resistance, and potential surface functionalization. Herein, we combined bioinspired membranes with in vitro assays to elicit the molecular mechanisms of gold shell-isolated nanoparticles (AuSHINs) on ductal mammary carcinoma cells (BT-474). Langmuir and Langmuir-Schaefer (LS) films were handled to build biomembranes from BT-474 lipid extract. AuSHINs incorporation led to surface pressure-area (π-A) isotherms expansion, increasing membrane flexibility. Fourier-transform infrared spectroscopy (FTIR) of LS multilayers revealed electrostatic AuSHINs interaction with head portions of BT-474 lipid extract, causing lipid chain disorganization. Limited AuSHINs insertion into monolayer contributed to hydroperoxidation of the unsaturated lipids upon irradiation, consistently with the surface area increments of ca. 2.0%. In fact, membrane disruption of irradiated BT-474 cells containing AuSHINs was confirmed by confocal microscopy and LDH leakage, with greater damage at 2.2 × 1013 AuSHINs/mL. Furthermore, the decrease in nuclei dimensions indicates cell death through photoinduced damage.

Ca2+ transient decline and myocardial relaxation are slowed during low flow ischemia in rat hearts.

The mechanisms that impair myocardial relaxation during ischemia are believed to involve abnormalities of calcium handling. However, there is little direct evidence to support this hypothesis. Therefore, we sought to determine whether the time constant of cytosolic calcium ([Ca2+]c) decline (tau Ca) was increased during low flow ischemia, and if there was a relationship between the time constant of left ventricular pressure decline (tau P) and tau Ca. Isolated perfused hearts were studied using indo-1 fluorescence ratio as an index of [Ca2+]c.tau P was used as an index of myocardial relaxation. The time constant of decline of the indo-1 ratio increased from 74 +/- 5 ms to 95 +/- 4, 144 +/- 10, and to 204 +/- 16 ms when coronary flow was reduced was reduced to 50, 20, and 10% of control, respectively. Indo-1 transients were calibrated to calculate tau Ca. tau Ca increased from 67 +/- 6 ms to 108 +/- 9 and 158 +/- 19 ms when coronary flow was reduced to 20 and 10% of control, respectively. There was a linear relationship between tau Ca and tau P (r = 0.82). These data support the hypothesis that during low flow ischemia, impaired myocardial relaxation may be caused by slowing of [Ca2+]c decline.

Relationship between myocardial metabolites and contractile abnormalities during graded regional ischemia. Phosphorus-31 nuclear magnetic resonance studies of porcine myocardium in vivo.

The mechanisms responsible for changes in myocardial contractility during regional ischemia are unknown. Since changes in high-energy phosphates during ischemia are sensitive to reductions in myocardial blood flow, it was hypothesized that myocardial function under steady-state conditions of graded regional ischemia is closely related to changes in myocardial high-energy phosphates. Therefore, phosphorus-31 nuclear magnetic resonance spectroscopy was employed in an in vivo porcine model of graded coronary stenosis. Simultaneous measurements of regional subendocardial blood flow, high-energy phosphates, pH, and myocardial segment shortening were made during various degrees of regional ischemia in which subendocardial blood flow was reduced by 16-94%. During mild reductions in myocardial blood flow (subendocardial blood flow = 83% of nonischemic myocardium), only the ratio of phosphocreatine to inorganic phosphate (PCr/Pi), Pi, and [H+] were significantly changed from control. PCr, ATP, and PCr/ATP were not significantly reduced from control with mild reductions in blood flow. Changes in myocardial segment shortening were most closely associated with changes in PCr/Pi (r = 0.94). Pi and [H+] were negatively correlated with segment shortening (r = -0.64 and -0.58, respectively) and increased over twofold when blood flow was reduced by 62%. Thus, these data demonstrate that PCr/Pi is sensitive to reductions in myocardial blood flow and closely correlates with changes in myocardial function. These data are also consistent with a role for Pi or H+ as inhibitors of myocardial contractility during ischemia.

Cardiac contractile dysfunction during mild coronary flow reductions is due to an altered calcium-pressure relationship in rat hearts.

Coronary artery stenosis or occlusion results in reduced coronary flow and myocardial contractile depression. At severe flow reductions, increased inorganic phosphate (Pi) and intracellular acidosis clearly play a role in contractile depression. However, during milder flow reductions the mechanism(s) underlying contractile depression are less clear. Previous perfused heart studies demonstrated no change of Pi or pH during mild flow reductions, suggesting that changes of intravascular pressure (garden hose effect) may be the mediator of this contractile depression. Others have reported conflicting results regarding another possible mediator of contractility, the cytosolic free calcium (Cai). To examine the respective roles of Cai, Pi, pH, and vascular pressure in regulating contractility during mild flow reductions, Indo-1 calcium fluorescence and 31P magnetic resonance spectroscopy measurements were performed on Langendorff-perfused rat hearts. Cai and diastolic calcium levels did not change during flow reductions to 50% of control. Pi demonstrated a close relationship with developed pressure and significantly increased from 2.5 +/- 0.3 to 4.2 +/- 0.4 mumol/g dry weight during a 25% flow reduction. pH was unchanged until a 50% flow reduction. Increasing vascular pressure to superphysiological levels resulted in further increases of developed pressure, with no change in Cai. These findings are consistent with the hypothesis that during mild coronary flow reductions, contractile depression is mediated by an altered relationship between Cai and pressure, rather than by decreased Cai. Furthermore, increased Pi and decreased intravascular pressure may be responsible for this altered calcium-pressure relationship during mild coronary flow reductions.

Role of MgADP in the development of diastolic dysfunction in the intact beating rat heart.

Sarcomere relaxation depends on dissociation of actin and myosin, which is regulated by a number of factors, including intracellular [MgATP] as well as MgATP hydrolysis products [MgADP] and inorganic phosphate [Pi], pHi, and cytosolic calcium concentration ([Ca2+]c). To distinguish the contribution of MgADP from the other regulators in the development of diastolic dysfunction, we used a strategy to increase free [MgADP] without changing [MgATP], [Pi], or pHi. This was achieved by applying a low dose of iodoacetamide to selectively inhibit the creatine kinase activity in isolated perfused rat hearts. [MgATP], [MgADP], [Pi], and [H+] were determined using 31P NMR spectroscopy. The [Ca2+]c and the glycolytic rate were also measured. We observed an approximately threefold increase in left ventricular end diastolic pressure (LVEDP) and 38% increase in the time constant of pressure decay (P < 0.05) in these hearts, indicating a significant impairment of diastolic function. The increase in LVEDP was closely related to the increase in free [MgADP]. Rate of glycolysis was not changed, and [Ca2+]c increased by 16%, which cannot explain the severity of diastolic dysfunction. Thus, our data indicate that MgADP contributes significantly to diastolic dysfunction, possibly by slowing the rate of cross-bridge cycling.

Thyroid hormone improves function and Ca2+ handling in pressure overload hypertrophy. Association with increased sarcoplasmic reticulum Ca2+-ATPase and alpha-myosin heavy chain in rat hearts.

We asked whether thyroid hormone (T4) would improve heart function in left ventricular hypertrophy (LVH) induced by pressure overload (aortic banding). After banding for 10-22 wk, rats were treated with T4 or saline for 10-14 d. Isovolumic LV pressure and cytosolic [Ca2+] (indo-1) were assessed in perfused hearts. Sarcoplasmic reticulum Ca2+-ATPase (SERCA), phospholamban, and alpha- and beta-myosin heavy chain (MHC) proteins were assayed in homogenates of myocytes isolated from the same hearts. Of 14 banded hearts treated with saline, 8 had compensated LVH with normal function (LVHcomp), whereas 6 had abnormal contraction, relaxation, and calcium handling (LVHdecomp). In contrast, banded animals treated with T4 had no myocardial dysfunction; these hearts had increased contractility, and faster relaxation and cytosolic [Ca2+] decline compared with LVHcomp and LVHdecomp. Myocytes from banded hearts treated with T4 were hypertrophied but had increased concentrations of alpha-MHC and SERCA proteins, similar to physiological hypertrophy induced by exercise. Thus thyroid hormone improves LV function and calcium handling in pressure overload hypertrophy, and these beneficial effects are related to changes in myocyte gene expression. Induction of physiological hypertrophy by thyroid hormone-like signaling might be a therapeutic strategy for treating cardiac dysfunction in pathological hypertrophy and heart failure.

Regulation of thyroid hormone receptor isoforms in physiological and pathological cardiac hypertrophy.

Physiological and pathological cardiac hypertrophy have directionally opposite changes in transcription of thyroid hormone (TH)-responsive genes, including alpha- and beta-myosin heavy chain (MyHC) and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), and TH treatment can reverse molecular and functional abnormalities in pathological hypertrophy, such as pressure overload. These findings suggest relative hypothyroidism in pathological hypertrophy, but serum levels of TH are usually normal. We studied the regulation of TH receptors (TRs) beta1, alpha1, and alpha2 in pathological and physiological rat cardiac hypertrophy models with hypothyroid- and hyperthyroid-like changes in the TH target genes, alpha- and beta-MyHC and SERCA. All 3 TR subtypes in myocytes were downregulated in 2 hypertrophy models with a hypothyroid-like mRNA phenotype, phenylephrine in culture and pressure overload in vivo. Myocyte TRbeta1 was upregulated in models with a hyperthyroid-like phenotype, TH (triiodothyronine, T3), in culture and exercise in vivo. In myocyte culture, TR overexpression, or excess T3, reversed the effects of phenylephrine on TH-responsive mRNAs and promoters. In addition, TR cotransfection and treatment with the TRbeta1-selective agonist GC-1 suggested different functional coupling of the TR isoforms, TRbeta1 to transcription of beta-MyHC, SERCA, and TRbeta1, and TRalpha1 to alpha-MyHC transcription and increased myocyte size. We conclude that TR isoforms have distinct regulation and function in rat cardiac myocytes. Changes in myocyte TR levels can explain in part the characteristic molecular phenotypes in physiological and pathological cardiac hypertrophy.

Nuclear magnetic resonance imaging-guided phosphorus-31 spectroscopy of the human heart.

Phosphorus-31 nuclear magnetic resonance spectroscopy can determine the status of high energy phosphates in vivo. However, its application to human cardiac studies requires precise spatial localization without significant contamination from other tissues. Using image-selected in-vivo spectroscopy (ISIS), a technique that allows three-dimensional localization of the volume of interest, 12 subjects were studied to determine the feasibility and reproducibility of phosphorus-31 spectroscopy of the human heart. Nuclear magnetic resonance imaging was performed using a commercial 1.5 tesla system to define the volume of interest. Phosphorus-31 spectra were obtained from the septum and anteroapical region of the left ventricle in 10 studies. Relative peak heights and areas were determined for high energy phosphates. The mean phosphocreatine to adenosine triphosphate ratio was 1.33 +/- 0.19 by height analysis and 1.23 +/- 0.27 by area analysis. Duplicate measurements in four subjects showed a reproducibility of less than or equal to 10% in three of the subjects. All spectra showed significant signal contribution from the 2,3 diphosphoglycerate in chamber red cells without evidence of skeletal muscle contamination. These results demonstrate the feasibility of image-guided phosphorus-31 spectroscopy for human cardiac studies and indicate the potential of this technique to study metabolic disturbances in human myocardial disease.

Substrate regulation of the nucleotide pool during regional ischaemia and reperfusion in an isolated rat heart preparation: a phosphorus-31 magnetic resonance spectroscopy analysis.

Isolated rat heart preparations were studied to characterise the alterations in high energy phosphates that occur during reversible regional ischaemia and to determine whether pyruvate, as the sole exogenous energy substrate, would attenuate the ischaemia induced depletion of the nucleotide pool when compared with glucose. Using phosphorus-31 magnetic resonance spectroscopy baseline concentrations of adenosine triphosphate, phosphocreatine, inorganic phosphate, and intracellular pH were compared with values during 30 min of left coronary artery occlusion followed by 30 min of reperfusion. These variables were related to changes in developed pressure, coronary flow, and oxygen consumption. In addition, the total nucleotide pool was evaluated by biochemical analysis of myocardial tissue extracts and coronary effluent. The ischaemic region was characterised by a dye staining technique and cross sectional echocardiographic measurements of regional myocardial wall thinning. In both glucose and pyruvate perfused groups, coronary flow and oxygen consumption decreased to 50-60% of control within 1 min of ischaemia and returned to baseline values with reflow. Developed pressure decreased to 50(9) and 74(8)% (mean(SEM] of control after 30 min of ischaemia in glucose and pyruvate perfused groups respectively. Reperfusion resulted in complete recovery of developed pressure in hearts perfused with pyruvate but not in the glucose group. Glucose perfused hearts had a greater decrease in intracellular pH during ischaemia (7.07(0.01) to 6.36(0.1] than pyruvate perfused hearts (7.06(0.02) to 6.83(0.04]. Reperfusion resulted in a rapid return to baseline intracellular pH in both groups. During ischaemia, adenosine triphosphate values decreased to a greater degree in glucose than in pyruvate perfused hearts (57(4) and 79(5)% of baseline respectively). Thirty minutes of reperfusion did not significantly improve adenosine triphosphate concentrations in either group. Phosphocreatine concentrations decreased to 52(7) and 75(6)% of baseline in glucose and pyruvate perfused groups respectively after the ischaemic period. Reperfusion resulted in normalisation of phosphocreatine values in the pyruvate but not in the glucose perfused group. Biochemical analysis of myocardial tissue extracts confirmed the spectroscopy data and showed that pyruvate inhibits the efflux of adenine nucleotide derivatives. Tissue concentrations of adenosine monophosphate were three times greater and adenosine 50% less after 30 min of ischaemia in the pyruvate perfused group.(ABSTRACT TRUNCATED AT 400 WORDS)

Basic mechanisms of myocardial dysfunction: cellular pathophysiology of heart failure.

The cellular pathophysiology of myocardial dysfunction in heart failure is multifactorial. Studies of animal models and myocardium from patients with heart failure have demonstrated abnormalities of cytosolic calcium handling, myofilament calcium sensitivity, and myocyte energetics. Many of these metabolic abnormalities have been shown to be the result of alterations in the activity or number of myocyte enzymes and transport channels that are important in excitation-contraction coupling. Several innovative techniques for measuring intracellular calcium and energy metabolites and recent advances in cell biology have helped to further our understanding of the cellular pathophysiology of heart failure. Abnormalities at several levels of the excitation-contraction coupling mechanism have been shown to be responsible for both systolic and diastolic dysfunction in the failing heart.

Basic mechanisms of myocardial dysfunction: cellular pathophysiology of heart failure.

Many studies published in 1994 significantly added to our understanding of the pathophysiology of heart failure at the cellular and subcellular level. This field continues to advance using different but complementary approaches. One approach is to study human myocardium, thereby providing data that is directly relevant to clinical disease. Another approach is to study mouse myocardium, taking advantage of transgenic technology to alter gene expression and directly study cause-and-effect relationships. Additionally, other animal models of heart failure (eg, pressure overload, volume overload, and paced tachycardia) continue to provide important information. Abnormalities of calcium cycling, myofilament sensitivity to calcium, cross-bridge kinetics, the myocyte cytoskeleton, and energetics have all been observed in animal models or failing human myocardium. The cellular and molecular basis for these abnormalities is now being explored. This understanding is essential for developing novel treatment strategies that may one day include gene therapy.

The dynamic structure of the germinal center.

Analysis of germinal centers (GCs) in chronically inflamed human tonsils has led to the dogma that GCs contain two compartments with separate functions: a dark zone where B cells proliferate and hypermutate; and a light zone where selection and differentiation occur. However, here Stephanie Camacho and colleagues discuss immunohistological analysis of splenic GCs arising de novo that reveal a more plastic structure.

Ca(2+)-dependent fluorescence transients and phosphate metabolism during low-flow ischemia in rat hearts.

To determine whether cytosolic free calcium ([Ca2+]i) rises during low-flow ischemia and to determine the mechanisms responsible for contractile dysfunction, isolated rat hearts were studied during graded reductions of coronary flow. Indo1 fluorescence at 385- and 456-nm wave-lengths (F385/456) was used as an index of [Ca2+]i. 31P-magnetic resonance spectroscopy (MRS) was used to measure free energy of ATP hydrolysis (delta GATP), intracellular pH (pHi), and Pi in parallel experiments to determine whether these factors may be responsible for increasing diastolic [Ca2+]i or altering the [Ca2+]i-pressure relationship. When coronary flow was reduced to 20 and 10% of control, diastolic F385/456 increased by 14 +/- 3 and 39 +/- 5%, respectively. Although developed pressure markedly decreased when coronary flow was reduced, there was no change of the F385/456 transient amplitude (systolic minus diastolic). During low-flow ischemia there was a significant decrease of delta GATP and increase of Pi that may lead to increased [Ca2+]i. Furthermore, there was a close inverse relationship between Pi and developed pressure, suggesting that Pi is an important regulator of contractility.

Quantitation of cytosolic [Ca2+] in whole perfused rat hearts using Indo-1 fluorometry.

Fluorometric determination of cytosolic calcium, [Ca2+]c, using Indo-1 in intact tissue, is limited by problems in obtaining calibration parameters for Indo-1 in vivo. Therefore, the goal of this study was to calibrate Indo-1 using in vitro constants, obtained from protein-containing reference solutions designed to produce similar Indo-1 spectral properties to those in vivo. Due to wavelength-dependent tissue light absorbance, the in vitro constants had to be absorbance-corrected using a novel method. The correction factor was calculated from the relationship between the Indo-1 fluorescence intensities at the two detection wavelengths. A mixture of proteins at approximately 28 mg/ml had a similar Indo-1 isosbestic wavelength (430 nm) to that found in vivo (427 nm), and a similar fluorescence ratio maximum with saturating Ca2+ to that found in vivo (after absorbance correction). Using calibration constants from this protein mixture, calculated [Ca2+]c in a Langendorf perfused rat heart was 187 nM during diastole, and 464 nM in systole. This new calibration method circumvented the considerable experimental problems of previous methods which required measurements with the cytosol fully depleted and fully saturated with Ca2+.

Investigation of factors affecting fluorometric quantitation of cytosolic [Ca2+] in perfused hearts.

The goal of these studies was to examine the effects of several factors that may artifactually influence quantitation of cytosolic [Ca2+], [Ca2+]c, while using the fluorescent calcium indicator Indo-1. The following factors were investigated: 1) a possible fluorescence contribution from unhydrolized Indo-1/AM (by Mn2+ quenching), 2) Ca2+ buffering by Indo-1 (by varying [Indo-1]), 3) endothelial and mitochondrial Indo-1 loading (by bradykinin stimulation and calculations), and 4) effects of changing tissue fluorescence (predominantly NAD(P)H) on calculated [Ca2+]c during hypoxia (by a new method which allowed simultaneous determination of [Ca2+]c and changes in [NAD(P)H]). No significant contribution of Indo-1/AM was found. With increasing [Indo-1], calculated systolic [Ca2+]c fell significantly. Indo-1 incorporation (< 18%) into endothelial cells, caused a slight underestimation of systolic [Ca2+]c, while mitochondrial Indo-1 loading may cause overestimation of [Ca2+]c. With increased tissue fluorescence, during hypoxia, systolic [Ca2+]c may be underestimated by approximately 27% (for Indo-1 loading factors three to five times original tissue fluorescence). These studies suggest conditions in which experimental artifacts could be minimized to allow reliable quantitation of [Ca2+]c in intact perfused hearts using Indo-1 fluorometry. The major problem of obtaining reliable results depended on the ability to correct for changing NAD(P)H fluorescence while keeping [Indo-1] low.

Protein and acidosis alter calcium-binding and fluorescence spectra of the calcium indicator indo-1.

The fluorescent indicator indo-1 is widely used to monitor intracellular calcium concentration. However, quantitation is limited by uncertain effects of the intracellular environment on indicator properties. The goal of this study was to determine the effects of protein and acidosis on the fluorescence spectra and calcium dissociation constant (Kd) of indo-1. With 350 nm excitation light, the ratio of indo-1 fluorescence in the absence versus the presence of saturating Ca2+ at wavelength lambda (S lambda) and Kd increased with [protein]. At pH 7.3, Kd, S400, and S470, which were 210 nM, 0.033, and 1.433 in the absence of protein, increased to 808 nM, 0.161, and 2.641, respectively, by adding proteins from frog muscle and to 638 nM, 0.304, and 3.039, respectively, by adding proteins from rat heart. Effects of protein on indo-1 fluorescence were reduced at higher [indo-1]. Acidosis (pH 6.3) had separate effects, which were additive to those of protein: in the absence of protein, acidosis increased Kd to 640 nM; frog muscle proteins further increased Kd to 1700 nM. Acidosis also changed S lambda slightly. In summary, interaction with protein or protons alters indo-1 calcium-binding and fluorescence. These findings are consistent with several previous studies and suggest that indo-1 calibration constants need to be derived in the presence of appropriate types of protein, ratio of [indo-1]/[protein], and pH.

Compensation for changes in tissue light absorption in fluorometry of hypoxic perfused rat hearts.

Fluorescence studies of NADH and various dyes in tissue are complicated by changes in light absorption, causing spurious results. Methods were developed to reduce and compensate for changes in light absorption in perfused rat hearts, subjected to normoxia and hypoxia. Isosbestic wavelengths were determined (i.e., absorption independent of oxygenation) in the whole ventricular wall and in the epicardium using transmitted and reflected light, respectively. Isosbestic wavelengths were found at approximately 385, 427, 455, 510, and 525 nm, similar in the epicardium, throughout the ventricle, in beating and arrested hearts, although the exact wavelengths varied among experiments. Furthermore, absolute light absorption was identical in the epicardium and endocardium. At nonisosbestic wavelengths, the effect of changing light absorption on fluorescence was quantified at various detection wavelengths using a reference dye. New correction methods were also developed and used to correct indo 1 fluorescence ratios for changing absorption so that results were independent of detection wavelengths. These methods can be used to greatly reduce artifacts due to changing tissue light absorption in a variety of fluorescence experiments.

Suppression of motion artifacts in fluorescence spectroscopy of perfused hearts.

Fluorescence spectroscopy of beating hearts has been used previously to measure intracellular regulators of function. Unfortunately, heart motion could introduce a spurious motion artifact (MA), influencing the measured fluorescence intensity ("signal"). To suppress MA, a ratio (or difference) has been calculated previously between the signal and an intensity reference detected at a different wavelength ("reference"). However, no studies have attempted to evaluate or optimize the efficiency of MA suppression. MA suppression was evaluated using reflected excitation light or fluorescence as reference. In addition, the MA contribution to the intensity ratio from a fluorescent dye, indo-1, was quantified. A reflected light reference resulted in poor suppression of MA. The use of a fluorescence reference resulted in suppression that was inversely related to the detection wavelength difference (delta) between the reference and signal. Therefore optimal MA suppression was obtained using a fluorescence reference at a wavelength close to the signal. For delta = 60 nm, MA was suppressed from approximately 10 to less than 2%. Finally, suppressed MA (delta = 60 nm) accounted for less than 10% of the indo-1 ratio fluctuations.