The effect of carbofuran-toxication on the chloragogen tissue of Tubifex tubifex Müll. (Oligochaeta).

Chloragogen cells, subserving ion exchange and electron accepting functions, were studied in Tubifex tubifex after insecticide treatment. Chloragogen cells were strongly influenced by in vivo carbofuran poisoning. The first alterations in the chloragogen cells became activated, both the formation and release of the chloragosomes reached a high rate. The released chloragosomes were phagocytosed by the amoebocytes. At an advanced stage of the toxication a heavy loading of the apical cytoplasm of chloragogen cells with lipid droplets, finally degenerative changes both in the chloragogen cells and amoebocytes were observed. Possible mechanisms of the carbofuran toxication and of the protective function of chloragogen cells in T. tubifex are discussed.     

Uptake and long-time storage of natural and synthetic dyes by earthworm chloragocytes. In vivo and in vitro investigations

Cationic or anionic dyes adsorbed onto cellulose granulate were transported across the gut wall, bound to blood proteins, and accumulated by the chloragocytes. Solubility in water promoted accumulation. The dyes ended up mainly in the chloragosomes. Down to 20 μmol dye per litre soil water resulted in visible accumulation. Worms which after dye-exposure were kept dye-free for 5 months retained substantial amounts of dye in the chloragosomes. In vitro experiments indicate that the binding to chloragosomes of synthetic and natural phenolics is by ion exchange with calcium phosphate and with an uncharacterized matrix-bound calcium chelator, aided by hydrophobic interactions between the dye and constituents of the chloragosome matrix. The findings are relevant for the evaluation of the effects of constant or periodic soil contamination with industrial or agricultural organochemicals.     

The coelomocytes of the earthworm Lumbricus terrestris: morphology and phagocytic properties.

The coelomocytes of Lumbricus terrestris have been classified and described, based on Wright's stained preparations and on living cells. The five major categories consist of basophils, acidophils, neutrophils, granulocytes and chloragogen cells. Both the acidophil and chloragogen cell groups contain two subgroups. Granulocytes also exhibit heterogeneity with respect to staining properties of granules. Some possess acidophilic granules, some basophilic granules, and others contain both types. Granules of acidophils have been observed to be occasionally excreted from the cells. All cell types, with the exception of chloragogen cells, produce pseudopodia and are capable of phagocytosis, a vital component of the earthworm's immune response.     

Optimization of staining conditions for microalgae with three lipophilic dyes to reduce precipitation and fluorescence variability

When the fluorescence signal of a dye is being quantified, the staining protocol is an important factor in ensuring accuracy and reproducibility. Increasingly, lipophilic dyes are being used to quantify cellular lipids in microalgae. However, there is little discussion about the sensitivity of these dyes to staining conditions. To address this, microalgae were stained with either the lipophilic dyes often used for lipid quantification (Nile Red and BODIPY) or a lipophilic dye commonly used to stain neuronal cell membranes (DiO), and fluorescence was measured using flow cytometry. The concentration of the cells being stained was found not to affect the fluorescence. Conversely, the concentration of dye significantly affected the fluorescence intensity from either insufficient saturation of the cellular lipids or formation of dye precipitate. Precipitates of all three dyes were detected as events by flow cytometry and fluoresced at a similar intensity as the chlorophyll in the microalgae. Prevention of precipitate formation is, therefore, critical to ensure accurate fluorescence measurement with these dyes. It was also observed that the presence of organic solvents, such as acetone and dimethyl sulfoxide (DMSO), were not required to increase penetration of the dyes into cells and that the presence of these solvents resulted in increased cellular debris. Thus, staining conditions affected the fluorescence of all three lipophilic dyes, but Nile Red was found to have a stable fluorescence intensity that was unaffected by the broadest range of conditions and could be correlated to cellular lipid content.     

Evidence of the presence of extraperoxisomal catalase in chloragogen cells of the earthworm, Lumbricus terrestris L

The DAB reactivity of the midintestine of the earthworm, consisting of epithelial layer, muscle layer, and chloragogen tissue, was examined electron microscopically. Besides the mitochondrial membranes of the examined cell types and the hemoglobin content of the blood vessels and chloragogen cells, a considerable DAB reactivity was found in the whole cytosol of the chloragocytes. The DAB reaction of the cytosol was more intensive when incubation medium for catalase, less intensive when incubation medium for peroxidase, was used and did not occur when H2O2 was omitted. Cytosol of the chloragogen cells was isolated and preliminary assay of catalase and peroxidase activities was made. Cytosol samples showed moderate peroxidase activity, but catalase activity measured by the decomposition of hydrogen peroxide showed a very high rate. Catalase and peroxidase activities of the cytosol were heat-sensitive and might have been inhibited by azide and cyanide, respectively. Results prove the assumption that the intensive DAB reactivity of the chloragocyte cytosol is caused by its extraperoxisomal catalase content.     

Evidence of the presence of extraperoxisomal catalase in chloragogen cells of the earthworm,Lumbricus terrestris L.

Summary The DAB reactivity of the midintestine of the earthworm, consisting of epithelial layer, muscle layer, and chloragogen tissue, was examined electron microscopically. Besides the mitochondrial membranes of the examined cell types and the hemoglobin content of the blood vessels and chloragogen cells, a considerable DAB reactivity was found in the whole cytosol of the chloragocytes. The DAB reaction of the cytosol was more intensive when incubation medium for catalase, less intensive when incubation medium for peroxidase, was used and did not occur when H₂O₂ was omitted.Cytosol of the chloragogen cells was isolated and preliminary assay of catalase and peroxidase activities was made. Cytosol samples showed moderate peroxidase activity, but catalase activity measured by the decomposition of hydrogen peroxide showed a very high rate. Catalase and peroxidase activities of the cytosol were heat-sensitive and might have been inhibited by azide and cyanide, respectively. Results prove the assumption that the intensive DAB reactivity of the chloragocyte cytosol is caused by its extraperoxisomal catalase content.     

Structural basis of cation exchange, complex formation and redox properties in chloragosomes.

Components of chloragosomes isolated from Octolasium transpadanum ROSA were separated by fractional extraction. The chemical composition of the fractions was determined and their effect on the anion- and cation-binding capability and on the lipophilic property of chloragosomes was studied. The acid-alcoholic extract of chloragosomes contained, among others, metalloporphyrins with 2 or 4 free -COOH groups; the residuum consisted of basic proteins. The metalloporphyrins and phosphatides gained by extraction with chloroform-methanol neutralize the basic groups of the chloragosome proteins, thus, an excess of free acidic groups develops, which is responsible for the polyanionic nature and cation exchange of chloragosomes. The apolar groups of phosphatides and carotenoids are responsible for the lipophilic nature of chloragosomes. Under experimental circumstances both in vivo and in vitro, organic cations were bound to chloragosomes, partially by a cation-exchange mechanism and partially by formation of more stable complexes. A small amount of anions may be absorbed by the bound cations. The diluted acidic extract of chloragosomes predominantly contain organic cations, in the present study riboflavin, flavin nucle0tide and thiamine were determined. Chloragosomes, owing to their structure, form complexes of varying stability with anions, cations and hydrophilic compounds equally. This feature explains their capability of cumulating trophic and toxic substances, while their redox activity is explained by their metalloporphyrin, flavin, thiamine and carotene content.     

The interactions of phthalocyanines with stimulated and resting human peripheral blood mononuclear cells.

The interactions of two metal-free phthalocyanines [(H2Pc) and Solar Pc (with four peripherical groups: SO2N(CH2CH2OH)2)] and of one metal substituted dye (CoPc) with resting and stimulated human peripheral blood mononuclear cells (PBMC) were compared. The absorption, fluorescence, photoacoustic and EPR spectra of both resting cells and cells stimulated by phytohaemagglutinin, incubated in dimethyl sulfoxide (DMSO) with very low or 95% water content and with or without dye addition, were measured. The fate of the light absorbed by the samples was investigated. It is known that singlet oxygen production is crucial for photodynamic action of dyes. Thermal deactivation and luminescence emission compete with this process, so investigation of these alternative paths of sensitizer deactivation provides information about photodynamic action. The incorporation of the investigated dyes into cells and the perturbation of the cell structure caused by the dyes, the incubation solvent and the activator were investigated by comparing the spectral properties of PBMC before and after stimulation and incubation. Incubation of the cells for 1 h in a solution of Solar Pc in 99.5% aqueous DMSO, resulted in an efficient dye incorporation which was highly selective. Solar Pc being introduced much more efficiently into stimulated cells than into resting cells.     

Microbial decolorization and degradation of synthetic dyes: a review

The synthesis of dyes and pigments used in textiles and other industries generate the hazardous wastes. A dye is used to impart color to materials of which it becomes an integral part. The waste generated during the process and operation of the dyes commonly found to contain the inorganic and organic contaminant leading to the hazard to ecosystem and biodiversity causing impact on the environment. The amount of azo dyes concentration present in wastewater varied from lower to higher concentration that lead to color dye effluent causing toxicity to biological ecosystem. The physico-chemical treatment does not remove the color and dye compound concentration. The decolorization of the dye takes place either by adsorption on the microbial biomass or biodegradation by the cells. Bioremediation takes place by anaerobic and/or aerobic process. The anaerobic process converts dye in toxic amino compounds which on further treatment with aerobic reaction convert the intermediate into CO₂ biomass and inorganics. In the present review the decolorization and degradation of azo dyes by fungi, algae, yeast and bacteria have been cited along with the anaerobic to aerobic treatment processes. The factors affecting decolorization and biodegradation of azo dye compounds such as pH, temperature, dye concentration, effects of CO₂ and Nitrogen, agitation, effect of dye structure, electron donor and enzymes involved in microbial decolorization of azo dyes have been discussed. This paper will have the application for the decolorization and degradation of azo dye compound into environmental friendly compounds.     

Cytochemical observations of coelomocytes from the earthworm, Lumbricus terrestris.

Synopsis Coelomocytes of the earthworm,Lumbricus terrestris, were stained by cytochemical techniques to determine the biochemical composition of the seven different cell types and subtypes. The enzymes acid phosphatase and -glucuronidase are present in all types of coelomocytes, but are especially abundant in basophils and neutrophils; the differences in enzyme amounts correlate well with the differences in phagocytic activity of the various cell types. No peroxidase is present. The cytoplasmic basophilia of basophils is due primarily to ribonucleic acid. Basophils also contain large deposits of glycogen, with neutrophils and chloragogen cells containing somewhat lesser amounts. The predominant granules of the two types of acidophils and of granulocytes are composed of a basic protein and a neutral mucopolysaccharide or glycoprotein. A second granule population, present in low numbers in acidophils and granulocytes, but in larger numbers in basophils and neutrophils, is small in size and lipid-positive and may, in part, represent lysosomes.Lipid is especially abundant in the vesicles and granules of the two types of chloragogen cells. Some granules of chloragogen cells also contain ferrous and ferric iron and a substance with pseudoperoxidase activity. The cytoplasm contains protein, glycogen, and a neutral mucopolysaccharide. In addition, acid mucopolysaccharides are variably present in the cytoplasm of chloragogen cells, the only coelomocytes to contain this class of substances.     

Hydrophobic acridine dyes for fluorescence staining of mitochondria in living cells. 2. Comparison of staining of living and fixed Hela-cells with NAO and DPPAO

The hydrophobic fluorescence dyes NAO and DPPAO (see scheme of structural formulae) stain the mitochondria of living HeLa-cells. The trans-membrane potential favours the dye accumulation of the cation NAO and supports the hydrophobic interaction of the dye with the mitochondrial membrane lipids and proteins. The lecithin-like dye DPPAO is electrical neutral. Its binding to mitochondria of living cells is only caused by hydrophobic interaction. NAO and DPPAO stain also the mitochondria of glutaraldehyde fixed HeLa-cells in aqueous medium. Fluorescence staining occurs even after extraction of the lipids of the cell with acetone. We suppose that the dye accumulation in the mitochondria of the fixed cells is caused by the hydrophobic interaction between the dyes and the very hydrophobic mitochondrial lipids and proteins.     

Lysosomal origin of the chloragosomes in the chloragogenous tissue of the earthworm Eisenia foetida: cytochemical demonstration of acid phosphatase activity

Summary The cytochemical localization of the lysosomal marker enzyme acid phosphatase was studied in the chloragogenous tissue of earthworms. The Gomori lead technique and the cerium capture technique were utilized. Both techniques demonstrated the chloragosomal location of this enzyme. Only a small proportion of chloragosomes presented reactivity, which suggests that these organelles are distinctly heterogeneous. The reaction product was localized in the periphery of chloragosomes, suggesting a membrane-bound compartmentalization of acid phosphatase. In addition, degenerating mitochondria and membrane whorls were observed in some chloragosomes, indicating the possibility that these organelles perform autophagosomal functions.     

The effect of lead incorporation on the elemental composition of earthworm (Annelida, Oligochaeta) chloragosome granules

The elemental compositions of chloragosome 'granules' in the earthworm Lumbricus rubellus living in non-polluted (Dinas Powys) and heavily Pb-polluted (Wemyss) soils were determined by fully quantitative electron probe X-ray microanalysis. P, Ca, S and Zn were the major elemental components of the chloragosomes. High Pb concentrations were found in chloragosomes of Wemyss animals; Pb was not detected in chloragosomes of Dinas Powys animals. Partial correlation and regression analysis indicated that the in vivo accumulation of Pb by chloragosomes was accompanied by diminished chloragosomal Ca concentrations. Pb is bound by P-containing ligand(s) in the chloragosome matrix. The sequestration of Pb by chloragosomes results in the detoxification of the metal by accumulative immobilization.     

A quantitative resolution of the spectra of a membrane potential indicator,diS-C3-(5), bound to cell components and to red blood cells

Summary Cationic cyanine dyes have been widely used to measure electrical potentials of red blood cells and other membrane preparations. A quantitative analysis of the binding of the most extensively studied of these dyes, diS-C₃-(5), to red blood cells and their constituents is presented here. Absorption spectra were recorded for the dye in suspensions of isolated red cell membranes and in solutions of cell lysate. The dependence of the spectra on the concentrations of dye and cell constituents shows that the dye binds to these membranes as monomers with an absorbance maximum at 670 nm instead of 650 nm as for free aqueous dye and that the dye binds to oxyhaemoglobin partly as monomer but primarily as dimer, with absorbance maxima ca. 670 and 595 nm, respectively. Quantitative estimates are derived for all binding constants and extinction coefficients. These estimates are applied to suspensions of whole cells to predict the dye binding, absorbance spectra, and calibration curves of binding and fluorescencevs. membrane voltage. Satisfactory agreement is found with binding and absorbance data for whole cells at zero membrane potential and with the binding and fluorescence data reported by Hladky and Rink (J. Physiol. (London) 263:287, 1976) for cells driven to positive and negative potentials using valinomycin. The marked tendency of oxyhaemoglobin to bind dye as dimer is not shared by some other proteins tested, including deocyhaemoglobin and oxymyoglobin.     

Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates.

Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coefficients. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, especially at high degrees of labeling. The results from our flow cytometry, immunocytochemistry, and immunohistochemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications.     

The Biological Stain Commission's Quality Control Laboratory operations and improved traceability of certified stains

Abstract

The Biological Stain Commission (BSC) is a quality control laboratory that certifies biological dyes for staining cells and tissues. Originally, a single lot of a certified dye was sold to histologists. Today, companies frequently change their lot numbers as part of regulatory efforts. When a certified dye undergoes a lot number change, the BSC must re-certify this dye to verify that it is identical to the one certified earlier. The BSC has improved how these lot changes are monitored using a redesigned BSC certification label. Certification labels always have been issued by the BSC and are attached to every bottle of “BSC certified dye” that is sold. The new BSC certification label has added security features and currently bears both the BSC certification number and the manufacturer batch lot number. The result is improved security and traceability of certified dyes.     

七甲川花菁染料作为肿瘤靶向和光动力治疗载体的研究进展

七甲川花菁近红外荧光染料(NIRF)可直接被肿瘤细胞特异性吸收,具有肿瘤靶向性。与化疗药物偶联后,该类染料可通过血脑屏障将药物转运至肿瘤部位,不仅可以减少化疗药物使用剂量,降低药物的毒副作用,也可通过近红外荧光成像实现对肿瘤治疗的实时监控。七甲川花菁染料所展示的线粒体毒性和光敏特性,可直接杀死肿瘤细胞,抑制肿瘤新生血管的形成。通过纳米包裹,能够显著增强该类染料的肿瘤靶向能力,实现实时跟踪药物释放情况。七甲川花菁染料特异性识别肿瘤细胞的能力与有机阴离子转运肽的作用密切相关,缺氧和线粒体膜电位也参与了染料吸收的调控。这些发现有利于将近红外荧光染料应用于肿瘤的靶向治疗。     

An integrated process of textile dye removal and hydrogen evolution using cyanobacterium,Phormidium valderianum

The work deals with the removal of textile dyes from wastewater using cyanobacteria and integrating the dye removal ability of the organism with the ability to produce hydrogen. Phormidium valderianum, a marine cyanobacterium, has been shown to remove more than 90% of textile dyes Acid red, Acid red 119 and Direct black 155 from the solutions in the pH range higher than 11. Presence of phenolic compounds and metal chelators drastically reduced the dye adsorption capacity of the organisms. The mechanism involved in the dye adsorption has been investigated. Hydrogen production by cells grown in presence of dyes in any phase of their growth was found to be less in comparison to that of control (grown without dye). A laboratory scale reactor was designed to integrate the hydrogen production and dye removal ability of P. valderianum.     

Species-specific effects on the optical signals of voltage-sensitive dyes

Summary The absorption changes of two merocyanine dyes in response to membrane potential changes were measured on several nueronal preparations to see whether the dyes would be useful in recording from these cells.We were able to record large signals without averaging from barnacle and leech neurons. The greatest signal with WW375 was seen at 750 nm. Much smaller increases in transmitted light intensity were seen at all other wavelengths between 500 and 780 nm. In contrast, vertebrate neuronal preparations produced much smaller signals with an entirely different action spectrum. Essentially the same spectrum was seen in cells of the sympathetic ganglion of the bullfrog,Rana catesbiana, dissociated chick spinal cord neurons, or dissociated rat superior cervical ganglion neurons. In each case an action potential was accompanied by increases in transmitted light intensity between 500 and 600 nm and 730 and 780 nm, and decreases in intensity between 600 and 730 nm with the dye WW375, the best dye tested. Similar results were obtained with dye NK2367 on both vertebrate and invertebrate preparations, except that the spectral properties were shifted 30 nm towards the blue. Both dyes caused some photodynamic damage to the cultured neurons after a few minute's exposure to the illuminating light. Several analogues of these dyes were also tried, but did not produce larger signals.     

Phototoxicity of the fluorescent membrane dyes PKH2 and PKH26 on the human hematopoietic KG1a progenitor cell line.

BACKGROUND: The phototoxic effects of the well-known fluorescent membrane dyes PKH2 and PKH26 have been unknown, although their use in cell tracking experiments has increased dramatically. To eliminate the phototoxicity-induced alteration in cell function and morphology, it is essential to examine the suspicious phototoxicity of these dyes. METHODS: Chemical and phototoxic effects of PKH dyes on the human hematopoietic KG1a cell line were examined. To minimize phototoxicity in long-term cell tracking experiments lasting up to 18 h with a fluorescence microscope system, time-lapse monitoring with different time intervals and exposure times was introduced. RESULTS: There were no significant effects of the two PKH dyes on cell viability and growth when using dye concentrations up to 5 microM. However, when stained cells were exposed to excitation light, cell viability decreased dramatically, showing the phototoxicity of the PKH dyes. More than 60% of cells stained with 5 microM PKH26 died after 5 min of continuous light exposure. The phototoxic effect was more extensive in cells stained with higher concentrations of the dyes. CONCLUSIONS: We present guidelines for the optimal use of these dyes by using a defined hardware configuration.