Oxalate pretreatment and use of a physical developer render the Kossa method selective and sensitive for calcium

Summary Based on experiments on agarose gels and tissue, a procedure has been developed which greatly improves the sensitivity and the specifity of the Kossa method for demonstrating calcium in tissue. Tissue calcium is immobilized by acetonic oxalic acid, which simultaneously removes the other sorts of anions capable of precipitating silver ions (e.g. phosphate, carbonate). The resulting submicroscopic grains of calcium oxalate are converted first into silver oxalate then into metallic silver by a treatment with silver nitrate followed by an ultra-violet irradiation (Kossa reaction). These submicroscopic metallic silver grains are enlarged up to microscopic visibility by means of physical development, which makes the staining highly sensitive. Costaining of the argyrophil sites in the tissue is totally suppressed by various tricks, which render the silver staining selective for calcium.     

A new method for the preparation of a calcium activated neutral protease highly sensitive to calcium ions

A Ca²⁺--activated neutral protease has been purified from chicken skeletal muscle to homogeneity by a new method which employs affinity chromatography on casein CH-Sepharose 4B. SDS polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single polypeptide chain with a molecular weight of 76,000. For half-maximum activity this protease requires 50 μM Ca²⁺ ions and its optimum pH is 7.6. The protease is inhibited by leupeptin, antipain, E-64 and endogenous inhibitor. The purified protease is very labile upon storage; after 3 days at 4°C no detectable activity remained.     

The von Kossa reaction for calcium deposits: silver lactate staining increases sensitivity and reduces background

Summary The classical von Kossa method has been modified: the high silver nitrate concentration in the original was replaced by 0.05% silver lactate with hydroquinone remaining the reducing agent of choice. The present modification stained calcification nodules with a sensitivity comparable to the original von Kossa reaction, but resulted in a reduced background staining in cultured osteoblasts. The method works well also with plastic- or paraffin-embedded tissue sections.     

Sugar-quantum dot conjugates for a selective and sensitive detection of lectins

One-pot synthesized neoglycoconjugates with a reactive thiol group are introduced here for functionalization with carbohydrates for solubilization and stabilization of CdSe-ZnS quantum dots in aqueous solution. Three different sizes of quantum dots (QDs) with lactose, melibiose, and maltotriose on their surface have been utilized, for the first time, for lectin detection through agglutination assay. The sugar-QDs thus synthesized were characterized by transmission election microscopy (TEM), fluorescence, and absorption spectroscopy. Agglutination of sugar-QDs by three different lectins occurred through specific multivalent carbohydrate-lectin interactions and was studied extensively by monitoring the scattered light at 600 nm. This assay was very selective, which has been demonstrated by a more selective binding of soybean agglutinin (SBA) with melibiose-QD, as compared to lactose-QD, and specific deagglutination caused by alpha-d-galactose, while alpha-d-mannose did not show any effect. The detection sensitivity of the maltotriose-QD was tested with Concanavalin A (ConA), and as little as 100 nM of the lectin was detected using light scattering. The detection sensitivity of this protocol has been enhanced considerably by the fluorescence properties of QDs. This agglutination process seems to occur through formation of smaller soluble aggregates, which further associate to form larger aggregates.     

Highly sensitive and selective derivatization-LC method for biomolecules based on fluorescence interactions and fluorous separations

A fluorescence derivatization LC method is a powerful tool for the analysis with high sensitivity and selectivity of biological compounds. In this review, we introduce new types of fluorescence derivatization LC analysis methods. These are (1) detection-selective derivatization methods based on fluorescence interactions generated from fluorescently labeled analytes: excimer fluorescence derivatization and fluorescence resonance energy transfer (FRET) derivatization; (2) separation-selective derivatization methods using the fluorous separation technique: fluorous derivatization, F-trap fluorescence derivatization, and fluorous scavenging derivatization (FSD).     

A new selective ultrahistochemical method for the demonstration of calcium using N,N-naphthaloylhydroxylamine

Summary A new, simple, rapid, and highly sensitive and selective method for the ultrahistochemical detection of calcium is described. The reagent N,N-Naphthaloylhydroxylamine (1,8-C₁₀H₆CON(ONa)CO) sodium salt was employed in this study for the demonstration of calcium at the subcellular level in relaxed and contracted muscles (smooth muscle of the stomach, thoracic aorta, and myocardial muscle cells) of the rat (in vitro as well as in vivo) and in the human vascular smooth muscle of the aorta with atherosclerotic calcification.Direct evidence of the presence of calcium in the electron-dense reaction products (calcium N,N-Naphthaloylhydroxylamine) is given by X-ray microanalysis of 1,500–2,000 Å thick sections.The significance of distributional differences in the localization of calcium in subcellular structures of relaxed and contracted muscles in discussed in relation to the role of calcium in the control of the muscle activity during the contraction-relaxation cycle.     

Development of a sensitive and selective method for the quantitative analysis of cortisol,cortisone, prednisolone and prednisone in human plasma

A highly selective, sensitive and robust LC–MS/MS method was developed for the simultaneous quantification of cortisol, cortisone, prednisolone and prednisone in human plasma. Prednisolone, cortisol and cortisone have similar fragmentation pattern. These three compounds were chromatographically separated, thus eliminating the inherent interference that fragments derived from the M + 2 and M isotopes of prednisolone contribute in the MRM channels of cortisol and cortisone, respectively. Additionally, by using a small particle (1.8 μm) analytical column, interferences present in the plasma samples from post-transplant recipients were successfully resolved from cortisol after a simple extraction consisting of protein precipitation, evaporation and reconstitution. The chromatographic separation was achieved on a Zorbax-SB Phenyl column under isocratic conditions during a run time of 8 min. Intra-run and inter-run precision and accuracy within ±15% were achieved during a 3-run validation for quality control samples at five concentration levels in charcoal-stripped plasma as well as in normal plasma, over a 500-fold dynamic concentration range. The lower limit of quantitation was 0.500 ng/mL for cortisone and prednisone, 1.00 ng/mL for cortisol and 2.00 ng/mL for prednisolone. The performance of the small particle column was maintained during more than 1200 injections in terms of peak retention time, symmetry and backpressure.     

Determination of nimodipine in human plasma by a sensitive and selective liquid chromatography-tandem mass spectrometry method

A sensitive and highly selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed to determine nimodipine in human plasma. The analyte and internal standard nitrendipine were extracted from plasma samples by n-hexane-dichloromethane-isopropanol (300:150:4, v/v/v), and chromatographed on a C(18) column. The mobile phase consisted of methanol-water-formic acid (80:20:1, v/v/v). Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI) source. The method has a limit of quantification of 0.24 ng/ml. The linear calibration curves were obtained in the concentration range of 0.24-80 ng/ml. The intra- and inter-day precisions were lower than 4.4% in terms of relative standard deviation (R.S.D.), and the accuracy ranged from 0.0 to 5.8% in terms of relative error (RE). This validated method was successfully applied for the evaluation of pharmacokinetic profiles of nimodipine tablets administered to 18 healthy volunteers.     

Extended sensitivity for the calcium selective electrode

Sensitivity of calcium-selective electrodes heretofore has been limited to calcium concentrations above 10(-8) M in the absence of competing ions. We describe the use of calcium buffers to stabilize the free calcium in the reference electrode. Electrode calibration is linear to 10(-8) M and is curvilinear to 10(-11) M in the presence of 0.1 M ionic strength. Selectivity with respect to competing cations, magnesium, potassium, sodium, and hydrogen is preserved. Electrode response time is less than 2 s for small changes in calcium activity. Response range is linear over 9 log units of calcium activity. Potential-time stability is less than 10 mV/h at saturation currents. Although the silver-silver chloride terminals are photosensitive throughout the visible and near-ultraviolet regions, housing the reference and indifferent in opaque barrels avoids false photovoltaic response.     

Calcium oxalate and the von Kossa method with reference to the influence of citric acid.

Crystals of calcium oxalate in pathological material and artificially precipitated in the absence or presence of citric acid in gelatin models were examined by polarized light and treated with silver nitrate under a variety of conditions. It was found that the intensity of the staining reaction was largely proportional to the strength of the silver solution, and was enhanced in in vitro studies by the presence of citric acid. Staining was also influenced by the crystal form, which was itself related to the absence or presence of citric acid.     

The combined use of isolated strips of guinea-pig lung parenchyma and ileum as a sensitive and selective bioassay for leukotriene B4

The biological effects of leukotriene (LT)B4 were compared, on a molar basis, with those of LTC4, LTD4, LTE4, 5-hydroxyeicosatetraenoic acid (5-HETE), PGD2, PGE1, PGE2, PGF2 alpha, PGI2, 6-oxo-PGF1 alpha, bradykinin (BK) and angiotensin II (Ang II) on isolated strips of guinea-pig lung parenchyma (GPP) and ileum smooth muscle (GPISM) superfused in series. LTB4 was similar to LTC4 and LTD4 on GPP, in relation to potency and contractions induced, but differed from LTE4 in being ten times more active and causing contractions of a much shorter duration of action on this tissue. However, unlike the other LTs, LTB4 produced contractions which were resistant to FPL 55712 (1.9 microM) and, when given repeatedly, caused tachyphylaxis in GPP. LTB4 was considerably more active on GPP than the other substances investigated. Further, PGD2, PGF2 alpha and PGI2 contracted GPP, the order of potency being PGD2 greater than PGF2 alpha approximately equal to PGI2, whereas PGE1 and PGE2 relaxed this tissue. In contrast to all other agonists tested which contracted GPISM, LTD4 displaying the highest activity, LTB4 was inactive on this tissue. 5-HETE and 6-oxo-PGF1 alpha were inactive on both GPP and GPISM. On the basis of differential effects of LTB4 on GPP and GPISM, this assay represents a simple and selective means to distinguish LTB4-like materials from other naturally-occurring substances likely to be generated in inflammatory fluids.     

A new selective ultrahistochemical method for the demonstration of calcium using N,N-naphthaloylhydroxylamine.

A new, simple, rapid, and highly sensitive and selective method for the ultrahistochemical detection of calcium is described. The reagent N,N-Naphthaloylhydroxylamine (1,8-C10H6CON(ONa)CO) sodium salt was employed in this study for the demonstration of calcium at the subcellular level in relaxed and contracted muscles (smooth muscle of the stomach, thoracic aorta, and myocardial muscle cells) of the rat (in vitro as well as in vivo) and in the human vascular smooth muscle of the aorta with atherosclerotic calcification. Direct evidence of the presence of calcium in the electron-dense reaction products (calcium N,N-Naphthaloylhydroxylamine) is given by X-ray microanalysis of 1,500-2,000 A thick sections. The significance of distributional differences in the localization of calcium in subcellular structures of relaxed and contracted muscles is discussed in relation to the role of calcium in the control of the muscle activity during the contraction-relaxation cycle.     

Activation of the human red cell calcium ATPase by calcium pretreatment

Some kinetic parameters of the human red cell Ca²⁺-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V _max of about 0.4 μmol P_i/mg × hr and a K _s of 0.3 μm Ca²⁺ were found. When Mg²⁺ (10 mm) or Ca²⁺(10 μm) were also added during preincubation, V _maxbut not Kwas altered. Ca²⁺ was more effective than Mg²⁺, thus increasing V _max to about 1.3 μmol P_i/mg × hr. The presence of both Ca²⁺ and Mg²⁺ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V _max. Conversely, addition of ATP (2 mm) with either Ca²⁺ or Ca²⁺ plus Mg²⁺increased V_max without affecting K. Preincubation with Ca²⁺ for periods longer than 30 min further increased V_maxand reduced Kto levels as low as found with calmodulin treatment. The Ca²⁺ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg²⁺, Ca²⁺ or Ca²⁺ plus Mg²⁺ did not differ significantly from each other. Moreover, immunodetection of Ca²⁺-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca²⁺ plus ATP in the presence of Mg²⁺ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca²⁺ effect. Ca²⁺ preincubation may stimulate the Ca²⁺-ATPase activity by stabilizing or promoting the E₁ conformation.     

Development of a selective and sensitive polymerase chain reaction assay for the detection of Mycoplasma pirum

Abstract A new assay using the polymerase chain reaction to amplify a 173-nucleotide DNA fragment within the 16S ribosomal RNA gene of Mycoplasma pirum has been developed. The assay selectively amplified DNA from all strains of M. pirum tested with a high level of sensitivity, even in a context of human DNA. DNA from other mollicute species, including those closely related to M. pirum , from bacteria phylogenetically close to mollicutes ( Clostridium innocuum, C. ramosum and Bacillus subtilis ), from Escherichia coli and from human peripheral blood mononuclear cells, did not produce the amplified DNA product specific for M. pirum .     

Rapid and sensitive method for prenatal diagnosis of propionic acidemia using stable isotope dilution gas chromatography-mass spectrometry and urease pretreatment

Propionic acidemia is one of the most frequent inborn errors of metabolism caused by a deficiency of propionyl-CoA carboxylase. Methylcitric acid, a key indicator of this disorder, is increased in amniotic fluid when a fetus is affected. Therefore, the direct chemical analysis of cell-free amniotic fluid for methylcitric acid, using stable isotope dilution gas chromatography-mass spectrometry, was carried out for the prenatal diagnosis of propionic acidemia. We developed a simple, highly sensitive, and accurate method for quantitation of this polar methylcitric acid in amniotic fluids by applying a simplified urease pretreatment which we devised earlier for urine. As the recovery of methylcitric acid from amniotic fluid was as high as 91% with a coefficient of variation lower than 3% in this procedure, only 0.02 ml of sample was required for the analysis of the affected fetus. This new procedure takes 1 h for sample pretreatment, including derivatization, and 15 min for GC-MS measurement and provides final results within 1.5 h.     

Double jeopardy: both overexpression and suppression of a redox-activated plant mitogen-activated protein kinase render tobacco plants ozone sensitive

In plants, the role of mitogen-activated protein kinase (MAPK) in reactive oxygen species (ROS)-based signal transduction processes is elusive. Despite the fact that ROS can induce MAPK activation, no direct genetic evidence has linked ROS-induced MAPK activation with the hypersensitive response, a form of programmed cell death. In tobacco, the major ROS-induced MAPK is salicylate-induced protein kinase (SIPK). We found through gain-of-function and loss-of-function approaches that both overexpression and RNA interference-based suppression of SIPK render the plant sensitive to ROS stress. Transgenic lines overexpressing a nonphosphorylatable version of SIPK were not ROS sensitive. Analysis of the MAPK activation profiles in ROS-stressed transgenic and wild-type plants revealed a striking interplay between SIPK and another MAPK (wound-induced protein kinase [WIPK]) in the different kinotypes. During continuous ozone exposure, abnormally prolonged activation of SIPK was seen in the SIPK-overexpression genotype, without WIPK activation, whereas strong and stable activation of WIPK was observed in the SIPK-suppressed lines. Thus, one role of activated SIPK in tobacco cells upon ROS stimulation appears to be control of the inactivation of WIPK.     

A sensitive cell-based method to screen for selective inhibitors of SMS1 or SMS2 using HPLC and a fluorescent substrate

Recent studies have revealed that sphingomyelin (SM) is involved in metabolic syndrome and is a new target of an anti-metabolic syndrome drug. Deficiencies in the enzyme SM synthase 1 (SMS1) result in severe abnormalities, whereas deficiencies in SMS2 do not. SMS1 and SMS2 synthesize SM under similar conditions, so their respective activities cannot be measured separately. We report here on a sensitive, high-throughput and reliable cell-based method to separately measure each SMS activity and to screen for SMS-specific inhibitors, using HPLC and fluorescent ceramide (Cer) analogs. We isolated SMS-null cells and stably transfected them with SMS1 or SMS2. Using these cells, individual SMS activities could be measured separately. Fluorescent Cer, SM, and glucosylceramide analogs could be separated within 4 min by HPLC using an NH₂ column. SMS activities of SMS1- or SMS2-expressing cells seeded in a single well of a 96-well plate could be measured using HPLC and fluorescent Cer analogs. This method clearly demonstrated that treatment of the cells with their respective siRNA or D609, an inhibitor of SMS, resulted in a significant decrease in each SMS activity. These results indicate that our newly developed method can be utilized for screening therapeutics against metabolic syndrome that target SMS2.