In vitro differentiation of rabbit blastocyst cells

Summary Six- and seven-day post-coitus (p.c.) rabbit embryos have been cultured in an attempt to establish a trophectodermal cell line. Results indicate that cells with epithelial characteristics (i.e. positive staining for cytokeratin) will survive in culture until Passage 3. At that time a fibroblastlike cell becomes predominant. In addition, we have found that the presence of the inner cell mass is required for embryo explants often results in the development of cells that spontaneously contract. These cells stain positively for myosin, which indicates that they may be precardiac cells. Maximum diastolic potential was −59±1.2 mV and the threshold potential was −53±2.3 mV. Spontaneously contracting cells did not respond to atropine, acetylcholine, epinephrine, isoproterenol, or propranolol. Action potential seems to be a result of an inward calcium current, because the beating rate is decreased in a dose-related manner with the calcium channel blocker verapamil, whereas the voltage-sensitive sodium channel blocker tetrodotoxin was without effect. This work was supported by grants HD21302, HD07069, DK31091, and HL37320 from the National Institutes of Health, Bethesda, MD, with additional support from a University of Alabama at Birmingham Cardviovascular Research and Training Center Award.     

Encephalic ventricles of the ewe (Ovis aries L.): conformation, relations and stereotaxic topography

Fifteen ewes, aged 3-6 years, were used for studying the conformation of the cerebral ventricles on mouldings, and the topography on scannographs and frozen cuts. The lateral ventricles show important variations, but the topography of their olfactory recess is constant.     

Acid-sensing ion channels in malignant gliomas

High grade glioma cells derived from patient biopsies express an amiloride-sensitive sodium conductance that has properties attributed to the human brain sodium channel family, also known as acid-sensing ion channels (ASICs). This amiloride-sensitive conductance was not detected in cells obtained from normal brain tissue or low grade or benign tumors. Differential gene profiling data showed that ASIC1 and ASIC2 mRNA were present in normal and low grade tumor cells. Although ASIC1 was present in all of the high grade glial cells examined, ASIC2 mRNA was detected in less than half. The main purpose of our work was to examine the molecular mechanisms that may underlie the constitutively activated sodium currents present in high grade glioma cells. Our results show that 1) gain-of-function mutations of ASIC1 were not present in a number of freshly resected and cultured high grade gliomas, 2) syntaxin 1A inhibited ASIC currents only when ASIC1 and ASIC2 were co-expressed, and 3) the inhibition of ASIC currents by syntaxin 1A had an absolute requirement for either gamma- or delta-hENaC. Transfection of cultured cells originally derived from high grade gliomas (U87-MG and SK-MG1) with ASIC2 abolished basal amiloride-sensitive sodium conductance; this inhibition was reversed by dialysis of the cell interior with Munc-18, a syntaxin-binding protein that typically blocks the interaction of syntaxin with other proteins. Thus, syntaxin 1A cannot inhibit Na(+) permeability in the absence of adequate plasma membrane ASIC2 expression, accounting for the observed functional expression of amiloride-sensitive currents in high grade glioma cells.     

Nongenomic regulation of ENaC by aldosterone

Aldosterone isinvolved in salt and water homeostasis. The main effect is thought toinvolve genomic mechanisms. However, the existence of plasma membranesteroid receptors has been postulated. We used whole cell patch clampto test the hypothesis that epithelial sodium channels (ENaC) expressedby renal collecting duct principal cells can be regulatednongenomically by aldosterone. In freshly isolated principal cells fromrabbit, aldosterone (100 nM) rapidly (<2 min) increased ENaC sodiumcurrent specifically. The aldosterone-activated current was completelyinhibited by amiloride. Aldosterone also activated ENaC in cellstreated with the mineralocorticoid receptor blocker spiranolactone.Nongenomic activation was inhibited by inclusion ofS-adenosyl-L-homocysteine in the pipettesolution, which inhibits methylation reactions. Also, the nongenomicactivation required 2 mM ATP supplementation in the pipette solution.Aldosterone did not activate any ENaC current in whole cell clamped ratcollecting duct principal cells. These functional studies areconsistent with aldosterone membrane binding studies, suggesting thepresence of a plasma membrane steroid receptor that affects cellularprocesses by mechanisms unrelated to altered gene expression.

     

gp120-induced alterations of human astrocyte function: Na(+)/H(+) exchange, K(+) conductance, and glutamate flux

Many human immunodeficiency virus(HIV)-infected patients suffer from impaired neurological function anddementia. This facet of the disease has been termed acquiredimmunodeficiency syndrome (AIDS)-associated dementia complex (ADC).Several cell types, including astrocytes and neurons, are notproductively infected by virus but are involved in ADC pathophysiology.Previous studies of rat astrocytes showed that an HIV coat protein(gp120) accelerated astrocyte Na⁺/H⁺ exchangeand that the resultant intracellular alkalinization activated apH-sensitive K⁺ conductance. The present experiments wereconducted to determine whether gp120 affected human astrocytes in thesame fashion. It was found that primary human astrocytes express apH-sensitive K⁺ conductance that was activated onintracellular alkalinization. Also, gp120 treatment of whole cellclamped human astrocytes activated this conductance specifically.Furthermore, gp120 inhibited glutamate uptake by primary humanastrocytes. These altered physiological processes could contribute topathophysiological changes in HIV-infected brains. Because thegp120-induced cell physiological changes were partially inhibited bydimethylamiloride (an inhibitor of Na⁺/H⁺exchange), our findings suggest that modification of human astrocyte Na⁺/H⁺ exchange activity may provide a means ofaddressing some of the neurological complications of HIV infection.

     

Participation of the chaperone Hsc70 in the trafficking and functional expression of ASIC2 in glioma cells

High-grade glioma cells express subunits of the ENaC/Deg superfamily, including members of ASIC subfamily. Our previous work has shown that glioma cells exhibit a basally active cation current, which is not present in low-grade tumor cells or normal astrocytes, and that can be blocked by amiloride. When ASIC2 is present within the channel complex in the plasma membrane, the channel is rendered non-functional because of inherent negative effectors that require ASIC2. We have previously shown that high-grade glioma cells functionally express this current because of the lack of ASIC2 in the plasma membrane. We now hypothesize that ASIC2 trafficking in glioma cells is regulated by a specific chaperone protein, namely Hsc70. Our results demonstrated that Hsc70 co-immunoprecipitates with ASIC2 and that it is overexpressed in glioma cells as compared with normal astrocytes. In contrast, there was no difference in the expression of calnexin, which also co-immunoprecipitates with ASIC2. In addition, glycerol and sodium 4-phenylbutyrate reduced the amount of Hsc70 expressed in glioma cells to levels found in normal astrocytes. Transfection of Hsc70 siRNA inhibited the constitutively activated amiloride-sensitive current, decreased migration, and increased ASIC2 surface expression in glioma cells. These results support an association between Hsc70 and ASIC2 that may underlie the increased retention of ASIC2 in the endoplasmic reticulum of glioma cells. The data also suggest that decreasing Hsc70 expression promotes reversion of a high-grade glioma cell to a more normal astrocytic phenotype.     

Photochemistry of dyes and fluorochromes used in biology and medicine: some physicochemical background and current applications

An overview of the basic principles of photochemistry is presented to facilitate discussion of fluorescence, quenching and quantum yields. These topics in turn provide the foundation for an account of fluorescence spectroscopy and its application to microscopy. A brief overview of light microscopy and the application of fluorescence microscopy is given. The influences of molecular features, such as aromatic character and substitution patterns, on color and fluorescence are described. The concept of color fading is considered with particular reference to its effect on microscopic preparations. A survey of representative fluorescent probes is provided, and their sensitivity, application, and limitations are described. The phototoxicity of fluorescent molecules is discussed using biomembranes and DNA as examples of targets of toxicity. Photodynamic therapy, a relatively new clinical application of phototoxicity, is described. Both anticancer and antimicrobial applications are noted, and an assessment is given of the current ideas on the ideal physicochemical properties of the sensitizing agents for such applications.