Double staining of immunoblot using enzyme histochemistry and India ink.

Immunoblotting is a commonly used technique for the immunodetection of specific proteins which have been fractionated by polyacrylamide gel electrophoresis. We describe here a simple procedure for the double staining of immunoblots, first to detect the immunoreactive component(s) by histochemistry using enzyme-conjugated secondary antibodies, and second to visualize the general protein electrophoretogram using India ink. This procedure permits the direct comparison of electrophoretic mobilities between the immunoreactive protein(s) and the total protein population as well as protein standards of known Mr. The experimental advantage of the procedure is that no additional manipulation of the protein samples or the standards is necessary prior to electrophoretic fractionation. In this report, detection of the vitamin D-dependent calcium-binding protein, calbindin-D28K, is used to illustrate the application of the procedure.     

Immobilization of catalytically active thromboxane synthase

Thromboxane synthase has been immobilized on phenyl-Sepharose beads by adsorption. The immobilized enzyme is catalytically active and has a slightly lower apparent Km for PGH2 than the detergent-solubilized enzyme. However, both imidazole- and pyridine-based inhibitors are equally effective in inhibiting the immobilized and solubilized enzyme preparations. Although the immobilized enzyme appears to be less stable than the solubilized enzyme it is sufficiently stable to be used as a model for studying the properties of the enzyme.     

Hybridization of biotinylated oligo(dT) for Eukaryotic mRNA quantitation

Quantitation of mRNA content in samples of total cellular RNA is required for the analysis of Northern blot hybridization to estimate the relative level of specific gene expression. Commonly used methods based on UV absorbance and dye staining measure only total RNA, and mRNA normalization by probing for mRNA levels of housekeeping genes, such as β-actin and glyceraldehyde-3-phosphate dehydrogenase, assumes a constant level of their expression, which, in fact, may vary as a function of cell proliferation and differentiation. We describe here a nonradioactive, slot-blotting method for quantifying eukaryotic mRNA levels using a biotinylated oligo(dT) probe, which hybridizes directly to the 3′-polyadenylated sequence of eukaryotic mRNAs. The method provides a more accurate estimation of mRNA content in total RNA samples and should be applicable for quantitative Northern analysis.     

Alterations in cellular calcium handling as a result of systemic calcium deficiency in the developing chick embryo: II. Ventricular myocytes.

We have previously shown that cardiovascular anomalies, such as hypertension and tachycardia, develop in Ca(2+)-deficient, shell-less (SL) chick embryos cultured ex ovo, accompanied by elevated circulating catecholamines and higher alpha-adrenergic sensitivity of cardiovascular functions. Results described in the preceding work, using erythrocytes as an experimental system, show that cellular Ca2+ handling properties are also altered as a result of long-term calcium deficiency. To examine the relevance of these findings to cells of the cardiovasculature, we have analyzed and compared the Ca2+ handling characteristics of the heart cells of SL and normal (NL) embryos. For this study, isolated and cultured ventricular myocytes of SL and NL embryos were loaded with Fura-2 via transient membrane damage with glass beads. Compared to Fura-2/AM, bead loading yielded similar values and kinetic profiles of [Ca2+]i-dependent differential fluorescence and, in addition, did not affect cell viability and beating activity. The Fura-2 loaded ventricular myocytes were washed in Ca(2+)-free buffer and then analyzed by ratiometric fluorescence (350 nm/380 nm) microscopy for kinetic changes in [Ca2+]i (R350/380 values) as a function of [Ca2+]o and adrenergic modifiers. At 0.5 and 1.0 mM [Ca2+]o, SL cells showed significantly higher [Ca2+]i, higher beating rates, and faster rate of increase in [Ca2+]i compared to NL cells. At higher [Ca2+]o (3.5 mM), there was no significant difference in [Ca2+]i and beating rate between NL and SL cells. Treatment with norepinephrine (NE; 0.01-1 microM) at 1 mM [Ca2+]o substantially increased [Ca2+]i in both NL and SL cells. In the former, the NE effect was completely inhibited by beta-blockade (1 microM propranolol). In contrast, in SL cells, NE remained effective after beta-blockade, and combined alpha-blockade (1 microM prazosin) and beta-blockade was needed to inhibit completely the NE effect. In both NL and SL cells, treatment with NE substantially increased beating rates in a similar manner. Taken together, these findings suggest that Ca2+ handling and adrenergic regulation of the heart cells are significantly altered in the SL embryos, and that these alterations may be related to the development of impaired cardiovascular functions resulting from systemic Ca2+ deficiency.     

Two different mechanisms of ampicillin resistance operating in strains of Vibrio cholerae O1 independent of resistance genes

Autoagglutinable strains of Vibrio cholerae O1 (seven nonfimbriate strains and one fimbriate strain) were transformed to obtain resistance to ampicillin. Two distinct mechanisms were found in these strains. One was operating in nonfimbriate strains by reducing OmpU protein production and the other was operating in a fimbriate strain (Bgd17) by newly overproducing cpxP protein. The twitching motility in the fimbriate Bgd17 strain disappeared depending on the production of cpxP protein, suggesting that fimbriation of V. cholerae O1 is controlled by a two-component signal transduction system.     

KDR activation is crucial for VEGF165-mediated Ca2+ mobilization in human umbilical vein endothelial cells

We have prepared apolyclonal mouse antibody directed against the first threeimmunoglobulin-like domains of the kinase insert domain-containingreceptor (KDR) tyrosine kinase. It possesses the ability to inhibitbinding of the 165-amino acid splice variant of vascular endothelialcell growth factor (VEGF₁₆₅) torecombinant KDR in vitro as well as to reduceVEGF₁₆₅ binding to human umbilical vein endothelial cells (HUVEC). These results confirm that the firstthree immunoglobulin-like domains of KDR are involved in VEGF₁₆₅ interactions. The anti-KDRantibody is able to completely blockVEGF₁₆₅-mediated intracellularCa²⁺ mobilization in HUVEC.Therefore, it appears that binding of VEGF₁₆₅ to the fms-like tyrosinekinase (Flt-1) in these cells does not translate into aCa²⁺ response. This is furtherexemplified by the lack of response to placental growth factor (PlGF),an Flt-1-specific ligand. Additionally, PlGF is unable to potentiatethe effects of submaximal concentrations ofVEGF₁₆₅. Surprisingly, theVEGF-PlGF heterodimer was also very inefficient at eliciting aCa²⁺ signaling event in HUVEC. Weconclude that KDR activation is crucial for mobilization ofintracellular Ca²⁺ in HUVEC inresponse to VEGF₁₆₅.

     

Serotonin and Inhibition in Limulus Lateral Eye

The response to light of one ommatidium is reduced or suppressed by simultaneous illumination of neighboring ommatidia. The mechanism of this lateral inhibition may be chemical synaptic transmission, based on the physiological findings of a number of investigators and on the following evidence. The fine structure of the neuropil of the lateral plexus exhibits numerous clear vesicles (ca. 400 A), dense-core vesicles (ca. 700–1400 A), Golgi regions, and other morphological features of neurochemical synapses. The indolealkylamine, serotonin (5-HT), even in nanomolar concentrations, has a potent inhibitory action. An initial, potent inhibitory dose of 5-HT produces a long lasting densensitization to subsequent doses. The desensitization affects lateral inhibition evoked by light stimulation of neighboring receptors, i.e. crossed-desensitization. Eye tissue extracts contain 5-HT and melatonin (MLT) at a level greater than 1 µg/g wet tissue and perhaps as high as 20–30 µg/g, as determined by two-dimensional thin-layer chromatography (TLC) and o-phthaldialdehyde fluorescence assay techniques. Subcellular fractionation on sucrose gradient indicates a peak in 5-HT and MLT content associated with an intermediate density fraction. 5-HT may be an inhibitory transmitter for lateral inhibition. One pathway for metabolism of 5-HT in the lateral eye may be via N-acetylserotonin and melatonin.     

Interfacial water on crystalline silica: a comparative molecular dynamics simulation study

Understanding the properties of interfacial water at solid–liquid interfaces is important in a wide range of applications. Molecular dynamics is becoming a widespread tool for this purpose. Unfortunately, however, the results of such studies are known to strongly depend on the selection of force fields. It is, therefore, of interest to assess the extent by which the implemented force fields can affect the predicted properties of interfacial water. Two silica surfaces, with low and high surface hydroxyl density, respectively, were simulated implementing four force fields. These force fields yield different orientation and flexibility of surface hydrogen atoms, and also different interaction potentials with water molecules. The properties for interfacial water were quantified by calculating contact angles, atomic density profiles, surface density distributions, hydrogen bond density profiles and residence times for water near the solid substrates. We found that at low surface density of hydroxyl groups, the force field strongly affects the predicted contact angle, while at high density of hydroxyl groups, water wets all surfaces considered. From a molecular-level point of view, our results show that the position and intensity of peaks observed from oxygen and hydrogen atomic density profiles are quite different when different force fields are implemented, even when the simulated contact angles are similar. Particularly, the surfaces simulated by the CLAYFF force field appear to attract water more strongly than those simulated by the Bródka and Zerda force field. It was found that the surface density distributions for water strongly depend on the orientation of surface hydrogen atoms. In all cases, we found an elevated number of hydrogen bonds formed between interfacial water molecules. The hydrogen bond density profile does not depend strongly on the force field implemented to simulate the substrate, suggesting that interfacial water assumes the necessary orientation to maximise the number of water–water hydrogen bonds irrespectively of surface properties. Conversely, the residence time for water molecules near the interface strongly depends on the force field and on the flexibility of surface hydroxyl groups. Specifically, water molecules reside for longer times at contact with rigid substrates with high density of hydroxyl groups. These results should be considered when comparisons between simulated and experimental data are attempted.