Influence of damaging and wilting red clover on lipid metabolism during ensiling and in vitro rumen incubation

This paper describes the relationship between protein-bound phenols in red clover, induced by different degrees of damaging before wilting and varying wilting duration, and in silo lipid metabolism. The ultimate effect of these changes on rumen biohydrogenation is the second focus of this paper. For this experiment, red clover, damaged to different degrees (not damaged (ND), crushing or frozen/thawing (FT)) before wilting (4 or 24 h) was ensiled. Different degrees of damaging and wilting duration lead to differences in polyphenol oxidase (PPO) activity, measured as increase in protein-bound phenols. Treatment effects on fatty acid (FA) content and composition, lipid fractions (free FAs, membrane lipids (ML) and neutral fraction) and lipolysis were further studied in the silage. In FT, red clover lipolysis was markedly lower in the first days after ensiling, but this largely disappeared after 60 days of ensiling, regardless of wilting duration. This suggests an inhibition of plant lipases in FT silages. After 60 days of ensiling no differences in lipid fractions could be found between any of the treatments and differences in lipolysis were caused by reduced FA proportions in ML of wilted FT red clover. Fresh, wilted (24 h) after damaging (ND or FT) and ensiled (4 or 60 days; wilted 24 h; ND or FT) red clover were also incubated in rumen fluid to study the biohydrogenation of C18:3n-3 and C18:2n-6 in vitro. Silages (both 60 days and to a lower degree 4 days) showed a lower biohydrogenation compared with fresh and wilted forages, regardless of damaging. This suggests that lipids in ensiled red clover were more protected, but this protection was not enhanced by a higher amount of protein-bound phenols in wilted FT compared with ND red clover. The reduction of rumen microbial biohydrogenation with duration of red clover ensiling seems in contrast to what is expected, namely a higher biohydrogenation when a higher amount of FFA is present. This merits further investigation in relation to strategies to activate PPO toward the embedding of lipids in phenol-protein complexes.     

Enzymatic removal of phenols from aqueous solutions by Coprinus cinereus peroxidase and hydrogen peroxide.

The fungal enzyme Coprinus cinereus peroxidase (CIP) can be used for the removal of toxic phenols from water. After treating aqueous solutions of phenols with CIP and H2O2 the phenols polymerized and precipitated. The decrease in phenol concentration was investigated for 10 different phenols. At neutral pH, the investigated phenols were in general removed with high efficiency.     

Fractionation and Phenol Composition of Cellulose Cigarette Smoke Condensate

Fractionation of smoke condensate and a detailed analysis of phenols in the cellulose cigarette smoke were performed during the course of systematic compositional studies of the smoke. Nearly equal yields of phenolic, acidic and neutral fractions were obtained in fractionation of cellulose cigarette smoke condensate. Thirtyseven phenols were newly identified in the smoke of cellulose cigarette in addition to six phenols ever found in pyrolysis products of cellulose. Semi-quantitative determination of some of these phenols revealed that dihydric phenols, such as catechol, hydroquinone, resorcinol and their alkyl derivatives were the main phenols and monohydric phenols were the minor phenols.     

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     

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.     

Induction of a white laccase from the deuteromycete Myrothecium verrucaria NF-05 and its potential in decolorization of dyes

Myrothecium verrucaria NF-05 is a deuteromycete fungus capable of producing a white laccase. The optimal concentration of Cu²⁺ for laccase production by this strain is 0.2 mM (43.23 ± 1.16 U mL^{? 1}). A comprehensive investigation of the induction demonstrated that NF-05 laccase production could be synergistically enhanced by various inducers, including aromatic phenols, amines and recalcitrant dyes, in the presence of 0.2 mM Cu²⁺. Sixteen phenols, fourteen amines and four dyes exhibited significant inductive effects on laccase production. The best inducer was 3, 3’-dimethylbenzidine, which increased laccase production to 258.1 ± 11.1 U mL^{? 1}. These results suggest that M. verrucaria NF-05 is a promising industrial laccase producer. Based on the increased production, purified NF-05 laccase was used to decolorize dyes of various structural types in the presence of six redox mediators. Among the 26 tested dyes, the decolorization rate of six azo dyes, chromotrope 2R, orange G6, Congo red, Ponceau S, amaranth and reactive yellow 135 and two arylmethane dyes, fast green 3 and neutral red, were significantly increased by each of the six mediators. These results demonstrate the potential use of the NF-05 laccase for the decolorization of recalcitrant dyes in dye bleaching and effluent detoxification.     

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.     

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.     

Histochemical Probing of Potato Periderm with Neutral Red: A Sensitive Cytofluorochrome for the Hydrophobic Domain of Suberin

A technique is described which uses the lipid fluorochrome neutral red as a cytochemical probe to detect the hydrophobic domain of the lignosuberin matrix in native and wound periderm of potato tuber. Toluidine blue O is used as a counterstain to quench autofluorescence. The neutral red technique appears to be specific for the hydrophobic/lipid domain of suberin and is significantly more sensitive than Sudan III and IV. The fluorochrome was extensively used on paraffin-embedded tissue with excellent results but also worked on freehand sections of fresh periderm tissue. In tuber tissue undergoing wound-healing, the pattern of suberin fluorescence obtained with the neutral red probe was identical in specificity to the color pattern obtained with Sudan III/IV, but somewhat different than that observed when berberine was used. Results obtained with the neutral red probe and berberine probe visually demonstrated that during ligno-suberin biosynthesis, the depositions of hydrophobic/lipid and phenolic/lignin-like components in potato tuber periderm were separate processes. The deposition of these components does not necessarily require their simultaneous presence because the fluorescence from these probes showed that the components were not consistently present together on the cell walls.     

Polyphenoloxidase from six mature grape varieties and their activities towards various phenols

Juices were prepared from three white and three red grape varieties harvested at full maturity and comparative studies on their oxygen-uptake, absorbance at 420 nm (degree of browning), polyphenoloxidase (EC 1.10.3.1; PPO) activity, and their phenol compositions were done. There was no correlation among the amounts of oxygen-uptake and oxidizable phenols in the juices and their degree of browning. However, there was similarity among the PPO from the six grape varieties in their general enzymatic properties and substrate specificity towards twenty-five phenols. A partially purified PPO fraction from Koshu juice, which did not contain free phenols, showed strong activity towards (+)-catechin, (−)-epicatechin, caffeic acid, catechol, pyrogallol, and protocatechuic acid (oxidizable phenols), but had no activity towards the other fifiteen phenols. The oxidizable substrates were not always the only limiting factor in the oxidation and browning of phenols by the PPO. Some unoxidizable phenols such as gallic acid, p-cresol, and tannic acid which were not substrates for PPO inhibited the oxidation of the oxidizable phenols except pyrogallol which was not inhibited by gallic acid. On the other hand, hydroquinone promoted the oxidation of the oxidizable phenols except protocatechuic acid. These showed that there were competitive reactions and synergism during the enzymatic oxidation of phenols.     

Effect of Photoperiod and End-of-Day Light Quality on Alkaloids and Phenolic Compounds of Tobacco

Tobacco plants (Nicotiana tabacum L.) were grown on long or short photoperiods followed by 5 minutes of red or far red radiation each day. Plants that received 16-hour photoperiods had a significantly higher concentration of total alkaloids and total phenolics than those that received 8-hour photoperiods. Significantly higher total alkaloid content was found in plants that received red rather than far red radiation last each day. Within each photoperiod, plants that received far red had higher concentrations of soluble phenols, particularly of chlorogenic acid. The interactions among these variables upon alkaloid and phenolic contents are discussed.     

Role of hydrophobic interactions and functional groups in the inhibitor binding to various sites of mitochondrial succinate-cytochrome c reductase

It was shown that the efficiency of succinate-cytochrome c reductase inhibitors, i. e. neutral polar substances, negatively charged phenols and 2-hydroxy-3-alkyl-1.4-naphthoquinones, is increased with an increase in their hydrophobicity. Plotting-lg C50 versus lg P for all the three groups of inhibitors, the role of functional groups of the inhibitors in their binding to the corresponding sites of the respiratory chain was determined. The efficiency of inhibition by neutral polar substances does not depend on the chemical nature of the inhibitors and is described by the equation-lg C50 = 0.864 lg P + 0.222 (r = 0.99). The negatively charged group of dissociated phenols determines the specificity of the inhibitor binding to the terminal site of the succinate dehydrogenase complex and is involved in the inhibitor binding to the enzyme. The carbonyl group of 2-hydroxy-3-alkyl-1.4-naphthoquinones selectively increases the affinity and efficiency of binding of these inhibitors to the b-c1 site of the respiratory chain.     

Semiquinone and ascorbyl radicals in the gut fluids of caterpillars measured with EPR spectrometry

The biological activity of phenolic compounds ingested by caterpillars is commonly believed to result from their oxidation, although the products of oxidation have been well-characterized in only a few cases. The initial oxidation products of phenols (semiquinone or phenoxyl radicals) can be measured with electron paramagnetic resonance (EPR) spectrometry. In this study semiquinone radicals formed from tannic acid and gallic acid in the gut fluids of two species of caterpillars were measured. In Orgyia leucostigma, in which ingested phenols are not oxidized, semiquinone radicals were absent or at very low intensities. By contrast, in Malacosoma disstria, in which ingested phenols are oxidized, high semiquinone radical intensities were measured. In the absence of detectable levels of semiquinone radicals, ascorbyl radicals were detected in the EPR spectra instead. High molar ratios of ascorbate to phenols in an artificial diet produced ascorbyl radicals in the midgut fluids of both species, while diets containing low molar ratios produced semiquinone radicals. Similar results were obtained in M. disstria fed the leaves of red oak or sugar maple. The results of this study provide further evidence that ascorbate is an essential antioxidant that prevents the oxidation of phenols in the gut fluids of caterpillars, and demonstrate that EPR spectrometry is a valuable method for determining the degree of oxidative activation of phenols ingested by herbivorous insects.     

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     

中性红再生紫膜的光化学性质

研究了中性红再生紫膜从先适应状态到暗适应状态的反应及再生紫膜中中性红的光吸收变化。实验结果说明紫膜上的金属离子结合位点可能深入膜内的质子通道,与构成质子通道的一些重要氨基酸残基相互作用。紫膜经去离子化处理变成蓝膜后,带有正电荷的质子化中性红也可以占据此金属离子结合位点,使蓝膜再生为紫膜。但金属离子与结合位点具有更强的亲和力,使蓝膜再生为紫膜的能力比质子化中性红强。     

In vitro studies of biological effects of cigarette smoke condensate. I. Genotoxic and cytotoxic effects of neutral, semivolatile constituents

Much of the biological activity of cigarette smoke resides in the neutral fraction of the particulate phase. Since the volatile constituents of this material, the semivolatiles, are accessible to selective filtration, some of the biological activity of cigarette smoke might be reduced. In view of this, the genotoxic and cytotoxic effects and the chemical composition of the semivolatile neutral material of a cigarette smoke condensate was investigated. Cigarette smoke condensate obtained from domestic American blend type cigarettes, was separated into a volatile, a nonvolatile and a semivolatile fraction. The semivolatile constituents were fractionated by liquid-liquid extraction into 4 subfractions: acids, phenols, bases and neutrals. The neutral material was separated further by silica gel chromatography into 7 subfractions of varying polarity. The major components of these were identified by gas chromatography-mass spectrometry. These fractions were studied using 4 in vitro short-term tests, of which 2, the Ames test and induction of sister-chromatid exchanges, provided information on their genotoxicity and the other 2 provided information on their cytotoxicity by measuring inhibition of cell growth and inhibition of oxidative metabolism. Sister-chromatid exchanges were induced by the neutral fraction and the 7 subfractions, the activities of which increased with increasing polarity. Neither the total neutral material, nor the subfractions showed any mutagenic activity in the Ames test. The cytotoxic effect of the fractions of medium polarity, was greater than that of the total neutral material, while the most and the least polar fractions were less toxic.     

Halide and Alkyl Phenols Block Volume-Sensitive Chloride Channels in Human Glial Cells (U-138MG)

Swelling of cells in hypotonic media activates a volume-sensitive Cl channel with well-known characteristics, but its structure and its regulation are still largely undetermined. It also has many inhibitors and most of them are also blocking other types of Cl channels. The numerous inhibitors of Cl channels have apparently no structural relationship among them. The purpose of this study was to try to determine the most simple molecules that could block these channels and identify some common properties among inhibitors. From the 37 new molecules that were studied, it was found that simple halide phenols like trichloro and triiodophenols could block these channels in the micromolar range. Also alkyl phenols, like butylphenols, are very sensitive blockers, comparable to other well-known blockers. But acidic halide phenols or nitrophenols are poor blockers. Also neutral polyphenols are more sensitive than acidic polyphenols. All these results indicate that the common basis for blocking these Cl channels is a phenol with hydrophobic groups, like short alkyl chains or an additional phenyl ring, attached to some of its sites, preferably sites 3-4-5. These results identify a new family of Cl channel blockers and hopefully improve our understanding of the blocking mechanism. Received: 28 August 1997/Revised: 12 December 1997     

遮荫和红膜处理对高山红景天根生物量及红景天甙含量季节变化的影响

采用纱布遮荫(覆以透明膜)和红色滤光膜处理人工种植生长3年和4年的高山红景天,观察了根生物量和红景天甙含量的季节变化结果表明,纱布遮荫处理(相对光强为全光照的51．8％)下根的生物量显著下降;红景天甙含量略有增加,红景天甙产量(红景天甙含量与根生物量乘积)则有所降低,但差异均不显著,红色滤光膜处理(相对光强为全光照的51．8％)与纱布遮荫处理相比,根生物量减少但不显著,红景天甙的含量和产量则显著增加,生长季末前者的红景天甙含量为后者的1．63倍(生长3年)和1．55倍(生长4年),红景天甙产量为后者的1．44倍(生长3年)和1．45倍(生长4年)。     

Spectrofluorometric studies of the lipid probe, nile red

We found that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, can be applied as a fluorescent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry (J. Cell. Biol. 1985. 100: 965-973). To understand the selectivity of the staining, we examined the fluorescence properties of nile red in the presence of organic solvents and model lipid systems. Nile red was found to be both very soluble and strongly fluorescent in organic solvents. The excitation and emission spectra of nile red shifted to shorter wavelengths with decreasing solvent polarity. However, the fluorescence of nile red was quenched in aqueous medium. Nile red was observed to fluoresce intensely in the presence of aqueous suspensions of phosphatidylcholine vesicles (excitation maximum: 549 nm; emission maximum: 628 nm). When neutral lipids such as triacylglycerols or cholesteryl esters were incorporated with phosphatidylcholine to form microemulsions, nile red fluorescence emission maxima shifted to shorter wavelengths. Serum lipoproteins also induced nile red fluorescence and produced spectral blue shifts. Nile red fluorescence was not observed in the presence of either immunoglobulin G or gelatin. These results demonstrate that nile red fluorescence accompanied by a spectral blue shift reflects the presence of nile red in a hydrophobic lipid environment and account for the selective detection of neutral lipid by the dye. Nile red thus serves as an excellent fluorescent lipid probe.