An investigation of hydroethidine as a fluorescent vital stain for prokaryotes

Abstract Hydroethidine (HE), manufactured by the chemical reduction of ethidium bromide (EB), was found to be decomposed to a bright red-fluorescent product by a range of microorganisms. Therefore, HE seemed to have potential as a fluorescent vital stain for microorganisms. However, inhibited or killed bacterial cultures still fluoresced red. Short periods (2–4 min) of excitation of sterile solutions of HE with a UV light yielded an orange/red product. Thus, HE seems to be decomposed biologically by a number of microorganisms but also abiotically by UV light-mediated process to red fluorescent material. These observations suggest that the oxidation of HE has only limited potential for assessing microbial activity.     

Uptake and accumulation of the herbicide bentazon by cultured plant cells

Cellular absorption of the herbicide bentazon, a weak acid with pK_a 3.45, was investigated using suspension-cultured cells of velvetleaf (Abutilon theophrasti Medic.). Bentazon accumulated rapidly to concentrations approximately four times that of the external medium. Bentazon accumulation against a concentration gradient was not due to its conversion to metabolites, partitioning into lipids, or binding onto cellular constituents. Bentazon uptake was related linearly to the external bentazon concentration, implying that movement of the herbicide into cells was not carrier-mediated. Bentazon was able to diffuse freely and extensively out of the cells, indicating that bentazon can readily diffuse across cell membranes. Potassium cyanide and carbonyl cyanide m-chlorophenyl hydrazone inhibited bentazon accumulation as did nitrogen gas when bubbled through the uptake medium. Absorption was pH-dependent with the greatest amount of bentazon accumulating at acidic external pH. Calculations indicated that conversion of uncharged bentazon to bentazon anion in the cytoplasm accounts for cellular accumulation of bentazon. These results provide evidence that bentazon is absorbed across membranes via simple diffusion and that bentazon accumulates in plant cells via an energy-dependent, ion-trapping mechanism which results in bentazon accumulation in the cytoplasm.     

Flow-cytometric detection of changes in the fluorescence emission spectrum of a vital DNA-specific dye in human tumour cells

A multiparameter flow cytometric technique has been used to detect changes in the emission spectrum of the DNA-specific fluorochrome Hoechst 33342 during uptake by intact, human tumour cells and during the in vitro titration of permeabilized cells. The spectral shift phenomenon was associated with changes in dye: DNA ratio revealing heterogeneity in dye-binding sites. The degree of spectral shift was sensitive to changes in pH within the physiological range. Surprisingly, chromatin structure, in terms of DNase accessibility, was not a major factor in the generation of the spectral shift. The technique of fluorescence emission analysis permits cells with similar DNA contents to be distinguished on the basis of changes in the microenvironment of chromatin for both fresh and freezer-stored biopsy or experimental preparations.     

Influence of trans-membrane potential and of hydrophobic interactions on dye accumulation in mitochondria of living cells

The lipophilic cationic fluorescent dye azopentylmethylindocarbocyanine (APMC) specifically stains the mitochondria in living cells. The dye contains a photosensitive diazirine ring and is suitable for photoaffinity labelling of mitochondrial proteins. By a combination of photoaffinity labelling of cell cultures of mouse fibroblasts (LM) with APMC, lysis of the labelled cells, subsequent micro-gel electrophoresis and detection of the fluorescence of the labelled proteins in the gel lanes with a sensitive microfluorimeter, we determined the number, apparent molecular masses, and relative intensity of the labelled proteins. In LM cells, three proteins with apparent molecular masses of 31, 40, and 74 kDa were labelled with high intensity, and proteins of 28, 29, 44, 48, 49, 66, and 105 kDa with low intensity. Two effects mainly determine the binding of lipophilic dye cations to mitochondrial proteins in living cells: (1) interaction of the trans-membrane potential of the inner mitochondrial membrane with the dye cations; and (2) hydrophobic interactions between the strongly lipophilic proteins of the inner membrane and the lipophilic dye molecules. Preincubation of the cell cultures with drugs that dissipate the trans-membrane potential, such as valinomycin, 2,4-dinitrophenol (DNP) and 3-chlorcarbonylcyanidephenylhydrazon (CCCP), strongly reduces or even prevents APMC labelling of mitochondrial proteins. The influence of hydrophobic interactions was investigated by competitive staining experiments using dyes with very different lipophilic properties. The lipophilicity of the dyes was characterized by their R _m values in reversed phase thin-layer chromatography. Prestaining with an excess of strongly lipophilic cationic acridine and phenanthridine dyes, such as pentyl acridinium orange chloride (PAO), nonyl acridinium orange chloride (NAO) and tetramethylpropidium chloride (MP), respectively, completely prevents protein labelling with APMC. Obviously, the dyes occupy the same mitochondrial binding sites as APMC. At equal concentrations the intensity of the 40-kDa signal is strongly reduced, whereas the 31-kDa and 74-kDa signals are unaffected. Using phenanthridine dyes with lower lipophilicity, namely propidium chloride (P), M, and N reduces the peak of the 40-kDa protein in APMC labelling, indicating that the 40-kDa protein preferentially binds lipophilic dye cations.     

Uptake of chylomicron remnants causes cholesterol accumulation in cultured human arterial smooth muscle cells

Chylomicron remnants (Sf greater than 100) were prepared by treating human chylomicrons (Sf greater than 400) with human post heparin plasma. Chylomicron remnants recovered after 70-80% of chylomicron triacylglycerol was hydrolyzed, suppressed LDL-receptor activity and increased cell cholesterol esterification to the same extent as did LDL when added to cultured human arterial smooth muscle cells at an equal cholesterol concentration. Cell cholesterol mass increased 36% after incubation with 25 micrograms LDL cholesterol/ml and 35% with 25 micrograms chylomicron-remnant cholesterol/ml. Addition of 30 microM chloroquine plus LDL or chylomicron remnants further increased cholesterol content of cells (74% and 87%, respectively) and caused a significant rise in cell esterified cholesterol (344% and 369%, respectively). Cholesterol content per unit of apolipoprotein B mass of remnants was 2-3-fold higher than that of LDL. Therefore, if lipoprotein particles were added at equivalent apolipoprotein B mass chylomicron remnants increased cell cholesterol content and cholesterol esterification and suppressed LDL receptor activity significantly more than did LDL. This suggests that an additional determinant, presumably apolipoprotein E, is important for receptor recognition of chylomicron remnants. These results may be relevant to the delivery of chylomicron-derived cholesterol to arterial cells proposed as a feature of atherogenesis.     

Visualization of Caenorhabditis elegans cuticular structures using the lipophilic vital dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal(1-4). The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility(4-6). Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer(7). Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development(8). Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages(2,9). Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology(5,6,10,11). While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression(12), antibody staining(13), and electron microscopy(1). Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures(14). Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail(15). We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.     

Hydroxyethyl lactamide, a dye solvent useful in vital staining.

In a search for new vital stains to reveal the microanatomy of nudibranch mollusks, the slow or very low solubility of many dyes in sea water posed a serious problem. Preliminary dissolution in tap water proved impractical. Hydroxyethyl lactamide, an odorless liquid and dye solvent was found ideal since it permits immediate attainment of saturated solutions of dyes in sea water. Since hydroxyethyl lactamide passed the severe "eolid nudibranch test" and has been found nonirritating for the very sensitive rhinophorial structures, and furthermore since it has been used by the pharmaceutical industry as a vehicle in antibiotic preparations, it appears to be an ideal universal dye solvent for general use in vital staining. It has been used extensively in unpublished research by the writer on vital staining of nudibranchs. It has a low order of physiological activity and can be regarded an essentially inert when used in vital staining.     

Growth of normal and neoplastic rat pleural mesothelial cells in the presence of conditioned medium from neoplastic mesothelial cells

Several transformed cells have been demonstrated to secrete growth factors. We studied the effect of conditioned medium from neoplastic rat pleural mesothelial cells on normal and neoplastic mesothelial cell growth. The results showed that the concentrated conditioned medium stimulated neoplastic mesothelial cell growth but inhibited reversibly normal mesothelial cell growth.     

Uptake and accumulation of exogenous docosahexaenoic acid by Chlorella

Tuna oil or its hydrolysate was added to a culture of Chlorella for its nutritional fortification as a feed for rotifer. Exogenous docosahexaenoic acid (DHA) in its free form was taken up by the cells of Chlorella vulgaris strain K-22 and by other strains, but tuna oil was not taken up by the cells. Accumulated DHA was found by electron microscopy in the cells in oil droplets. All strains of Chlorella used in these experiments took up exogenous DHA into the cells. It seems that the structure of the cell wall did not affect the uptake of DHA into the Chlorella cells.     

Uptake and activation of eicosapentaenoic acid are related to accumulation of triacylglycerol in Ramos cells dying from apoptosis

The present study investigates the mechanism behind induction of cell death by eicosapentaenoic acid (EPA) in leukemia cells. The PUFA-sensitive cell lines Raji and Ramos, which die by necrosis and apoptosis upon incubation with EPA respectively, had 2- to 3-fold higher uptake rate of EPA than the PUFA-resistant U-698 cell line. Furthermore, Ramos cells contained more lipid bodies and 3-fold more triacylglycerol than U-698 cells after 24 h incubation with 60 microm EPA. The mechanism behind the increased rate of EPA uptake in the PUFA-sensitive cell lines was examined by comparing the expression of 6 different fatty acid binding proteins (FABPs) and 3 acyl-CoA synthetases (ACSs) in U-698 and Ramos cells. Moreover, enzymatic activity of ACS and acyl-CoA:1,2-diacylglycerol acyltransferase (ADGAT) was investigated. The protein expression level of CD36 and p-FABPpm, the mRNA level of FABP, liver-FABP, heart-FABP, intestinal-FABP, ACS1, ACS2, and enzymatic ADGAT activity were similar in the two cell lines. However, an mRNA signal observed with a probe for ACS3 was 1.7 times higher in Ramos than in U-698 cells, and lysate from Ramos cells had a higher capacity to activate EPA to EPA-CoA than U-698 cell lysate.In conclusion, the present findings indicate that cellular uptake, activation and incorporation of EPA into lipids may be related to induction of cell death in leukemia cell lines.     

Differentiation and the neoplastic transformation of normal cells

It is suggested that blastomogenic factors can be considered as the means isolating normal cells from each other. Carcinogenic substances caused cell functions to disturb, to inhibit or to intensify the synthesis of specific substances, to change the cell surface disrupted contacts of interactions between cells, pressing cells to become on the way of evolution, and to create heterogenic populations of cells.     

Detection of neoplastic cells in the bloodstream]

A study was made of the efficacy of trypan blue, acridine orange, tetracycline and oxytetracycline for detection of tumour cells injected into the blood stream of rats. The cells were identified in the mesenteric microvessels by intravital microscopy. Fluorescence of fluorochromized cells was observed in the blue-violet (lambda max = 400 nm) and ultra-violet (lambda max = 365 nm) irradiation of the fluorescent lamp and in the laser irradiation (lambda = 337 nm). The cells stained with acridine orange had a higher fluorescence intensity and a more distinct structure than those labelled with tetracyclines. Identification of cells with trypan blue was more difficult. The fluorescent method of determination is rather simple and permits to indentify tumour cells directly in the blood stream.     

Uptake,accumulation and metabolism of auxins in tobacco leaf protoplasts

Uptake and metabolism of exogenous naphthalene-1-acetic acid (NAA) and indole-3-acetic acid (IAA) have been studied in tobacco (Nicotiana tabacum L. cv. Xanthi) mesophyll protoplasts. Both auxins entered protoplasts by diffusion under the action of the transmembrane pH gradient without any detectable participation of an influx carrier. Molecules were accumulated by an anion-trapping mechanism and most of them were metabolized within hours, essentially as glucose-ester and amino-acid conjugates. Protoplasts were equipped with a functional auxin-efflux carrier as evidenced by the inhibitory effect of naphthylphtalamic acid on IAA efflux. Basically, similar mechanisms of NAA and IAA uptake occurred in protoplasts. However, the two auxins differed in their levels of accumulation, due to different membrane-transport characteristics, and the nature of the metabolites produced. This shows the need to estimate the accumulation and the metabolism of auxins when analyzing their effects in a given cell system. The internal auxin concentration could be modulated by changing the transmembrane pH gradient, giving an interesting perspective for discriminating between the effects of intra- and extracellular auxin on physiological processes.Abbreviations BA benzoic acid - C_i/C_e accumulation ratio of auxin - IAAasp N-[3-indolylacetyl]-dl-aspartic acid - NAA naphthalene-1-acetic acid - NAAasp N-[1-naphthylacetyl]-l-aspartic acid - NPA N-1-naphthylphthalamic acid The authors thank Dr. M. Caboche (I.N.R.A, Versailles, France) for his generous gifts of some amide derivatives of 1-NAA, Mr. P. Varennes and Dr. B. Das (I.C.S.N., C.N.R.S., Gif-sur-Yvette, France) for recording and interpreting the mass spectra of NAA glucose ester, and Prof. P. Manigault (Institut des Sciences Végétales, Gif-sur-Yvette) for microscopy measurements of protoplast dimensions. This work was supported by funds from the C.N.R.S, I.N.R.A, and E.E.C.     

Uptake,accumulation and phytoextraction efficiency of cesium in Gypsophila paniculata

Abstract

To evaluate the phytoextraction efficiency of Gypsophila paniculata from Cs-contaminated soils and analyze the mechanism of Cs accumulation in G. paniculata, we analyzed the characteristics of Cs bioaccumulation and subcellular distribution, in addition to its chemical forms in the plant under hydroponic conditions. The results showed that total Cs content in the aboveground parts and the entire plant were as high as 6137.32?mg·kg^?1?dry weight and 7338.49?mg·kg^?1?dry weight, respectively, after 17?days in the 50?mg·L^?1 Cs treatment. The BCF was between 2.35 and 3.38. The TF was between 1.00 and 2.46 in G. paniculata. Subcellular distribution of Cs in the plant was as follows: soluble fraction?>?cell wall?>?organelles. Inorganic Cs (F-ethanol) and water-soluble Cs (F-dH2O) were the main types of Cs in G. paniculata. Further studies show that the phytoextraction efficiency can reach 10.30–11.91% planting a season of G. paniculata under potted conditions. The results suggested that G. paniculata, a perennial, drought-tolerant herb, was a high-accumulator of Cs, which may have potential uses in phytoremediation of Cs-contaminated soil.