中性红与去离子化紫膜（蓝膜）的相互作用

本文研究了带有正电荷的质子化中性红使去离子化紫膜（蓝膜）再生为紫膜的能力,以及再生紫膜的光化学性质,并从结合中性红的光谱特性中,探讨了结合位的微环境特点,实验结果说明,中性红具有使蓝膜再生为紫膜的能力,但再生紫膜与天然紫膜相比有较慢的光循环速率和较低的质子泵效率,结合至蓝膜上的中性红吸收最大值相对于自由中性红有明显的蓝移,说明了结合中性红是以双体形式存在于亲水环境中,文中讨论了紫膜上金属离子结合位     

Uptake and intracellular distribution of neutral red in cultured fibroblasts

Neutral red (2-methyl-3-amino-7-dimethylamino-phenazine) is taken up by cultured fibroblasts through a non-saturable process and its concentration in the cells reaches several hundred times that in the medium. The dye stains consistently discrete cytoplasmic granules; their size appears related to the level of cellular accumulation of neutral red. By isopycnic centrifugation of cytoplasmic extracts in sucrose gradients, we could clearly evidence an association of neutral red with (1) the lysosomal enzymes cathepsin D and N-acetyl-β-glucosaminidase; it is thought that neutral red accumulates in lysosomes by proton trapping; (2) cell constituents equilibrating at a median density of 1.15 g/cm³; this second compartment, with a concentration power as large as lysosomes, becomes apparent only when neutral red is more than 25 μM in the culture fluid; it serves as a temporary storage site, and the dye is thereafter transferred to lysosomes. We suggest this second compartment to be the Krinom vesicles, i.e. large autophagic vacuoles induced by and containing neutral red. Finally, a small amount of intracellular neutral red could be associated with either secretory or endocytic vesicles.     

The accumulation of neutral red in illuminated thylakoids

Thylakoids isolated from spinach (Spinacia oleracea L.) bind only a small fraction of neutral red in the dark whereas they accumulate large amounts of the protonated dye in their inner space under light. Light-induced neutral red uptake depends on the size of the proton gradient across the thylakoid membrane but does not follow the mechanism established for amines. Instead, the correlation between pH gradient and neutral red uptake can be predicted quantitatively assuming that protonated neutral red is accumulated mainly as dimer species.Under appropriate conditions, accumulation of protonated neutral red in the inner thylakoid space is proportional to an absorbance increase at 520 nm. This 520-nm change can be used for the continuous measurement of pH changes in thylakoids during steady-state illumination.     

Proton movements and electric potential generation in reconstituted ATPase proteoliposomes from the thermophilic cyanobacterium Synechococcus 6716

ATP hydrolysis-induced proton translocation and electric potential generation have been studied in ATPase proteoliposomes by means of various optical probes. The proteoliposomes consisted of reconstituted ATPase complex and native lipid mixture isolated from the thermophilic cyanobacterium Synechococcus 6716 [Van Walraven et al. (1983) Eur. J. Biochem. 137, 101-106]. The native cartenoids and added oxonol VI served as probes for the electric membrane potential generated by the net charge separation (negative outside, positive inside). Their responses, with similar half-times as 9-tetradecylamino-6-chloro-2-methoxyacridine, are sensitive to valinomycin and stimulated by nigericin, as expected. The proton concentrations of extraliposomal and intraliposomal aqueous spaces were monitored by neutral red and cresol red; for internal measurements these pH indicators were trapped inside the vesicles during detergent dialysis. Internal acidification and external alkalinization induced by ATP hydrolysis are inhibited by nigericin and enhanced by valinomycin; at the commonly used higher valinomycin concentrations the neutral red response becomes transient, while the much slower cresol red response is diminished right from its onset. At smaller preset pH gradients both ATP hydrolysis activity and neutral red response are diminished. At increasing MgCl2 concentrations the neutral red responses are slowed down and the cresol red responses are slightly enhanced; this is observed for both internal and external dye responses. Neutral red permeation through the membrane is insignificant under our experimental conditions but is enhanced at temperatures below the lipid-phase transition. In the case of externally added neutral red the non-permeant buffer Hepes is only effective at high MgCl2 concentration, whereas some external cresol red response is visible only at high MgCl2 concentration in the presence of Hepes. The kinetics of the pH indicator and electric potential probe responses clearly distinguish fast interfacial and intra-membrane proton displacements from slow bulk proton equilibration. The data are summarized in a model that supports the importance of localized proton displacements for the primary energy-transducing events.     

The buffering capacity of the internal phase of thylakoids and the magnitude of the pH changes inside under flashing light.

The buffering capacity inside thylakoids is determined and the magnitude of flash-induced pH changes inside is calibrated in the pH range from 6.4 to 8.1. The work is based on flash-induced absorption changes of neutral red in a chloroplast suspension in which the outer phase is strongly buffered by bovine serum albumin. It is shown that neutral red is bound inside thylakoids. The binding can be described by a simple isotherm with an apparent Km = 4 microM and satruation at 1 neutral red per 17 chlorophylls. The apparent pK of neutral red is shifted from 6.6 in solution to 7.25 when bound inside. It is demonstrated that neutral red is a clean indicator of pH changes inside, i.e. when properly used it shows no response to other events. Although bound it reports pH changes which occur in the internal osmolar (aqueous) volume of thylakoids. This is obvious from the influence of chemically very different buffers on the magnitude of the absorption changes of neutral red. These act in a manner proportional to their calculated buffering capacity in aqueous solution. The intrinsic buffering capacity of the internal phase is determined with the aid of these buffers, at pH 7.2 it is between 0.8 and 1 mM (at 60 mosM). The absence of large variations in the buffering capacity in the range from pH 6.4 to 8.1 suggests that proteinaceous groups are involved in addition to the lipids which may dominate the buffering capacity at lower pH. The magnitude of the internal pH change is arrpox. 0.6 (at pH 7.3) under stimulation of both photosystems with a short xenon flash of light.     

The Preparation of Vital Neutral Red

Neutral red iodide suitable for vital staining was prepared by condensing nitrosodimethylanilin hydrochloride with m-toluylenediamine and the indamine, toluylene blue, was obtained. This was subjected to air oxidation and converted to the eurhodine, neutral red. The purification of this dye was brought about by converting it into its comparatively insoluble stannous chloride double salt, filtering, dissolving in water, and precipitating the neutral red iodide with potassium iodide solution. This was re-dissolved in water, reprecipitated with potassium iodide solution and crystallized from 95% ethanol. The uncrystallized dye was also found satisfactory for vital staining. Several other preparations of neutral red iodide were made, using a somewhat different procedure than that given above, and it was generally found that satisfactory stains were obtained only when the preparation was free from toluylene blue.

The chloride of the color base was prepared by continuing the air oxidation of the toluylene blue until a test sample indicated its complete conversion into neutral red. The color was salted out with sodium chloride and crystallized from 95% ethanol. Both the crystallized and uncrystallized products were found to be excellent stains.     

The Preparation of Vital Neutral Red

Neutral red iodide suitable for vital staining was prepared by condensing nitrosodimethylanilin hydrochloride with m-toluylenediamine and the indamine, toluylene blue, was obtained. This was subjected to air oxidation and converted to the eurhodine, neutral red. The purification of this dye was brought about by converting it into its comparatively insoluble stannous chloride double salt, filtering, dissolving in water, and precipitating the neutral red iodide with potassium iodide solution. This was re-dissolved in water, reprecipitated with potassium iodide solution and crystallized from 95% ethanol. The uncrystallized dye was also found satisfactory for vital staining. Several other preparations of neutral red iodide were made, using a somewhat different procedure than that given above, and it was generally found that satisfactory stains were obtained only when the preparation was free from toluylene blue.

The chloride of the color base was prepared by continuing the air oxidation of the toluylene blue until a test sample indicated its complete conversion into neutral red. The color was salted out with sodium chloride and crystallized from 95% ethanol. Both the crystallized and uncrystallized products were found to be excellent stains.     

The buffering capacity of the internal phase of thylakoids and the magnitude of the pH changes inside under flashing light

The buffering capacity inside thylakoids is determined and the magnitude of flash-induced pH changes inside is calibrated in the pH range from 6.4 to 8.1. The work is based on flash-induced absorption changes of neutral red in a chloroplast suspension in which the outer phase is strongly buffered by bovine serum albumin. It is shown that neutral red is bound inside thylakoids. The binding can be described by a simple isotherm with an apparent K_m = 4 μM and saturation at 1 neutral red per 17 chlorophylls. The apparent pK of neutral red is shifted from 6.6 in solution to 7.25 when bound inside. It is demonstrated that neutral red is a clean indicator of pH changes inside, i.e. when properly used it shows no response to other events. Although bound it reports pH changes which occur in the internal osmolar (aqueous) volume of thylakoids. This is obvious from the influence of chemically very different buffers on the magnitude of the absorption changes of neutral red. These act in a manner proportional to their calculated buffering capacity in aqueous solution. The intrinsic buffering capacity of the internal phase is determined with the aid of these buffers, at pH 7.2 it is between 0.8 and 1 mM (at 60 mosM). The absence of large variations in the buffering capacity in the range from pH 6.4 to 8.1 suggests that proteinaceous groups are involved in addition to the lipids which may dominate the buffering capacity at lower pH. The magnitude of the internal pH change is approx. 0.6 (at pH 7.3) under stimulation of both photosystems with a short xenon flash of light.     

Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O

Nile red is a phenoxazone dye that fluoresces intensely, and in varying color, in organic solvents and hydrophobic lipids. However, the fluorescence is fully quenched in water. The dye acts, therefore, as a fluorescent hydrophobic probe. We utilized this novel property of nile red to develop a sensitive fluorescent histochemical stain for tissue lipids. Nile red was prepared by boiling Nile blue A under reflux for 2 hr in 0.5% H2SO4, and extracting the product into xylene. For staining, the purified dye is dissolved in 75% glycerol (1-5 micrograms/ml) and applied to frozen tissue sections. Tissue lipids then fluoresce yellow-gold to red, depending on their relative hydrophobicity. Using sections of liver and aorta from a cholesterol-fed rabbit, we assessed the value of Nile red as a stain for neutral lipids by comparing the staining pattern obtained with that produced by oil red O, a commonly used dye for tissue cholesteryl esters and triacylglycerols. In the cholesterol fatty liver, Nile red staining was comparable to that of oil red O. In contrast, Nile red staining of rabbit aortic atheroma revealed ubiquitous lipid deposits not observed with oil red O staining. These latter results suggest that Nile red can detect neutral lipid deposits, presumably unesterified cholesterol, not usually seen with oil red O or other traditional fat stains.     

Electron microscopic characterization of calcium-binding physodes in the green alga Mougeotia scalaris.

Effect of the covalently cross-linking agents glutardialdehyde and osmium tetroxide, and of adsorption of the vital dye, neutral red, to the matrix of the calcium-binding "vesicles" from the green alga Mougeotia scalaris has been analysed in situ, both in terms of structural preservation and of the calcium-binding capacity of the vesicles. Upon cell fixation in glutardialdehyde without OsO4, the vesicles appear to dissolve, but upon simultaneous fixation in glutardialdehyde with OsO4 (1% w/v), the vesicles retain a globular form, are evenly stained by osmium and appear to be surrounded by a membrane-like structure. This structure was also observed around the vesicles in cells preincubated for 10 min in 0.1 mM neutral red and then fixed in glutardialdehyde/OsO4 for 1 h. More detailed information of the matrix structure is obtained when simultaneous fixation of the Mougeotia cells was shortened to 15 min: a membrane-like structure was no longer observed around the vesicles. After cell treatment in the presence of neutral red, no calcium at all was found inside the vesicles. A small amount of calcium remained, when cells were fixed simultaneously and extensively in the absence of neutral red. However, calcium was found, to a considerable extent, inside the vesicles after short simultaneous fixation of the cells in the absence of neutral red. Based on the ultrastructural and elemental features presented here, the calcium-binding vesicles in Mougeotia appear to represent a member of the large family of (calcium-binding) physodes in lower plants (CaBP).     

Optimising a clearance index based on neutral red as an indicator of physiological stress for bivalves

Neutral red is a weakly cationic dye that is soluble in water, has a low toxicity for almost all classes of organisms, and has been used as a histological and vital stain since the early twentieth century. Estimating the volume of water cleared of indicator material by suspension feeding bivalves (clearance or filtration rate) was one of the earliest applications of neutral red; however, less than thirty studies have applied this methodology since it was first described in 1954. The feeding/clearance rate is used as a sensitive indicator of physiological stress and is therefore an early-warning tool that is suitable for monitoring the ecological status of water bodies. The aim of our study was to optimise a clearance index based on neutral red solution by addressing the effect of i) the acidifying pH; ii) the holding temperature before spectrophotometric reading; and the time iii) before and iv) after the acidification of solutions of neutral red used to carry out clearance assays. Furthermore, as a case study we fine-tuned the clearance assay for the edible estuarine bivalve, Cerastoderma edule. The results showed that there were no statistical differences as regards the absorbance of neutral red solutions holding at 4 or 20 °C or a solution acidified between the ranges of pH 4–5. However, the absorbance significantly decreased as the pH increased to pH 6. The time before acidification had no significant effect on absorbance. Once the neutral red solution is acidified, the absorbance decreases over time, signifying that the absorbance should be read in the first 24 h. The concentration of neutral red used in the experiences should be sufficient to allow final concentrations of over 0.5 mg/L after the clearance period, since we observed that the sensitivity of this protocol decreased at low concentrations. In the case of C. edule, the optimum clearance conditions per individual were found to be 100 ml of 4 mg/L of neutral red dye during a 30 min period in dark conditions. A bioassay using a clearance index of C. edule based on this simple colorimetric technique would appear to be a potential tool for implementation in environmental monitoring programmes for water quality assessment in accordance with European directives. We trust that the new harmonised protocol will become a widely used and cost-effective means to monitor the clearance index as an indicator of physiological stress for bivalves.     

尼罗红荧光法快速检测培养过程中湛江等鞭金藻的中性脂

研究一种快速准确测定微藻中中性脂的方法。湛江等鞭金藻是一种中性脂含量高且具有开发潜力的能源微藻。以湛江等鞭金藻为实验对象,首先优化尼罗红染色的条件。当二甲基亚砜体积分数为2.0%、尼罗红质量浓度为1.00μg/m L、细胞密度为1.0×106个/m L、激发波长为480 nm、检测波长为580 nm时,优化的染色时间为10min。其次测定了背景荧光对检测的影响。结果表明,在不同细胞状态下,背景荧光强度大约是微藻内荧光强度的20%左右,可以忽略。最后比较了尼罗红荧光法和重量法。结果表明,荧光强度与中性脂含量的相关系数R2=0.946 8,虽然两者相关性并不十分高,但作为一种快速测定微藻中中性脂的方法,尼罗红荧光法依然是研究微藻培养过程中中性脂含量变化的有效方法。     

Mycobacterium smegmatis displays the Mycobacterium tuberculosis virulence-related neutral red character when expressing the Rv0577 gene

Neutral red staining is a cytochemical reaction that has been found to be related to Mycobacterium tuberculosis virulence and, therefore, the component involved in it is thought to be a virulence factor. To study the molecular basis of this reaction we constructed an M. tuberculosis cosmid library in Mycobacterium smegmatis and selected recombinant neutral red positive clones. Heterologous complementation identified Rv0577 as the gene responsible for this trait and we have also shown that it is expressed as a single polycistronic unit together with Rv0576 which could also be involved in the neutral red staining.     

Enhancement of St. Louis Arbovirus Plaque Formation by Neutral Red

Experiments with St. Louis encephalitis (SLE) virus have shown that neutral red enhances the plaque size in duck embryo cell cultures. This may represent a new method for genetic studies of SLE virus population. In a mosquito pool specimen NR(+) and NR(-) particles were demonstrated. By intracerebral passage in mice, there is a selection of NR(+) particles. Similar effects were not shown for eastern, western, and California encephalitis viruses. Plaques formed by the latter virus, however, were significantly reduced in number by neutral red.     

The control of food mobilisation in seeds of Cucumis sativus L.

The degradation of neutral lipid and the development of lipase activity in cucumber cotyledons is stimulated by white light. Malate synthase and isocitrate lyase activities show no stimulation. Lipase activity and neutral lipid breakdown are also enhanced by red light, far-red light proving ineffective. Far-red light reverses the effect of red light indicating the involvement of phytochrome in the control of lipase activity. Although light stimulates neutral lipid degradation it appears that much of the additional lipid lost is used in the synthesis of polar lipid constituents. Furthermore, the influence of light on lipid degradation appears to be species dependent.Abbreviations WL white light - RL red light - FRL far-red light     

Selective vital staining of companion cells of potato tuber and parsnip root with neutral red.

When staining the internal phloem region of a potato tuber with the vital stain neutral red, it was observed that files of elongated cells of narrow diameter were heavily stained and were easily distinguishable from the more isodiametric parenchyma cells, many of which did not stain with neutral red. The elongated cells were identified as companion cells by locating the adjacent sieve-tube members through counterstaining with aniline blue and reviewing under violet light. Of a number of other plants surveyed, only parsnip roots possessed companion cells exhibiting a similar slective staining. In other plants both the companion cells and the surrounding parenchyma cells usually stained. Sieve-tube members never accumulated neutral red. It was concluded that the vacuoles of the companion cells of the potato tuber were stained by the ion trap mechanism because of the color of the accumulated stain, the lack of staining when neutral red was applied in an acidic solution, and the complete destaining after soaking in dilute ammonium hydroxide.     

Characterization of the isolated calcium-binding vesicles from the green alga Mougeotia scalaris,and their relevance to chloroplast movement

The calcium-binding vesicles from the green alga Mougeotia scalaris were isolated and characterized after staining in vivo by neutral red or rhodamine B. They were found to possess, a protonated group with a pK_a-9.9, typifying phenolic hydroxyl groups; upon titration, both, phenolic compound(s) and vital dye were concomitantly released from the vesicular matrix. A shift in peak absorbance from 450 nm to 540 nm of the vitally stained vesicles indicated that the neutral form of neutral red was bound to the vesicular, matrix as an intermediate form, stabilized via intermolecular hydrogen bonds to the phenolic compound(s). Up to 8.5.10⁹ dye molecules were calculated to be adsorbed to a mean-size vesicle. Analysis of Langmuir adsorption isotherms, indicated that there were two binding sites each for both neutral red and rhodamine B. The isolated vesicles were devoid of calcium, probably because vesicular calcium, bound to the vesicle matrix, was displaced upon dye binding. Dye adsorption to the vesicles in vivo results in substantial inhibition of the reorientational movement of the Mougeotia chloroplast and is explained by dye-mediated disorder of the cellular calcium homoeostasis.Abbreviations NR neutral red - RB rhodamine B - SDS sodium dodecyl sulfate This paper is part of the Ph.D. thesis of F. Grolig at Justus-Liebig-Universität Giessen, FRG     

Localized or delocalized protons in photophosphorylation? On the accessibility of the thylakoid lumen for ions and buffers

The deposition of protons inside thylakoids after flash excitation was measured photometrically with neutral red as pH indicator. In continuation of previous work (Junge, W., Ausländer, W., McGeer, A. and Runge, T. (1979) Biochim. Biophys. Acta 546, 121–141), we studied the influence of salts on neutral red binding and on the pK of the heterogeneous protonation-deprotonation of inside-bound neutral red as a function of salts. With freeze-thawed (cryoprotective dimethyl sulphoxide) or aged chloroplasts, we observed that the heterogeneous pK of inside-bound neutral red was salt dependent in a way which suggested that neutral red was bound close to the plane of negative fixed charges and that the adjacent inner aqueous phase could accommodate an extended ionic double layer. This, together with the known extremely rapid proton exchange between surface layer and adjacent bulk phase, led us to conclude that inside-deposited protons rapidly reached an aqueous inner bulk phase. This conclusion was corroborated by the observation that extremely hydrophilic buffers like phosphate quenched the transient internal acidification independent of whether proton deposition was due to water oxidation or to plastohydroquinone oxidation. Very different behaviour was observed for freshly prepared chloroplasts with broken outer envelope. Here, inside-bound neutral red was seemingly unaffected by salts and hydrophilic buffers failed to quench the internal acidification. The electrical conductivity and proton permeability of the thylakoid membrane, on the other hand, were as usual. We attributed the seeming inaccessibility of the internal phase to the failure to accommodate a sufficiently extended ionic cloud between the tightly appressed membranes. In such material we observed hindered lateral mobility of protons at the outer side of the thylakoid membrane. This was tentatively attributed to multiple binding-debinding at buffering groups. The consequences for the chemiosmotic theory are: There is one type of damaged chloroplast material, which is competent in photophosphorylation and where protons are deposited into an internal aqueous bulk phase in the orthodox sense. In more intact material, however, the internal space lacks the characteristic properties of an aqueous bulk phase and there is evidence for lateral diffusion limitation for protons. Here, the thermodynamics of photophosphorylation may be inadequately described by the proton-motive force between two aqueous phases which are each isopotential.     

Selective Vital Staining of Companion Cells of Potato Tuber and Parsnip Root with Neutral Red

When staining the internal phloem region of a potato tuber with the vital stain neutral red, it was observed that files of elongated cells of narrow diameter were heavily stained nod were easily distinguishable from the more isodiametric parenchyma cells, many of which did not stain with neutral red. The elongated cells were identified as companion cells by locating the adjacent sieve-tube members through counterstaining with aniline blue and viewing under violet light. Of a numb of other plants surveyed, only parsnip roots possessed companion cells exhibiting a similar selective staining. In other plants both the companion cells and the surrounding parenchyma cells usually stained. Sieve-tube members never accumulated neutral red. It was concluded that the vacuoles of the companion cells of the potato tuber were stained by the ion trap mechanism because of the color of the accumulated stain, the lack of staining when neutral red was applied in an acidic solution, and the complete destaining after waking in dilute ammonium hydroxide.     

Neutral red uptake assay for the estimation of cell viability/cytotoxicity

The neutral red uptake assay provides a quantitative estimation of the number of viable cells in a culture. It is one of the most used cytotoxicity tests with many biomedical and environmental applications. It is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in the lysosomes. Most primary cells and cell lines from diverse origin may be successfully used. Cells are seeded in 96-well tissue culture plates and are treated for the appropriate period. The plates are then incubated for 2 h with a medium containing neutral red. The cells are subsequently washed, the dye is extracted in each well and the absorbance is read using a spectrophotometer. The procedure is cheaper and more sensitive than other cytotoxicity tests (tetrazolium salts, enzyme leakage or protein content). Once the cells have been treated, the assay can be completed in <3 h.