Effective pH pretreatment and cell disruption method for real-time intracellular enzyme activity assay of a marine fungus covered with pigments

Filamentous fungi are capable producers of many bioactive compounds, and real-time intracellular enzyme activity assay is an essential guidance for their bioprocess developments. However, there are many difficulties in preparing homogenate for enzyme activity assay, such as disrupting fungal cell with complicated cellular structure and solid cell wall, removing abundant extracellular metabolites accumulating on mycelia, and so on. Halorosellinia sp. (No. 1403) was a marine-derived filamentous fungus producing a potential antitumor compound 1403C, and the deep red pigments (with main component of 1403C) covering on its mycelia showed strong absorption in a wide range, which critically affected the measurement of many enzyme activities. In this study, we developed an effective pH pretreatment and cell disruption method to prepare homogenate for enzyme activity assay. When mycelia were washed by the solution with pH 5.0 for 3?min, most pigments could be removed without severe loss on enzyme activities. Afterward, grinding with mini bead for 15?min with alternating cooling could effectively disrupt both cell wall and mitochondrial membrane. These methods have been successfully applied on real-time intracellular enzyme activity assay of Halorosellinia sp. (No. 1403) and can offer enlightenment for other filamentous fungi with similar problems.     

A new study of cell disruption to release recombinant thermostable enzyme from Escherichia coli by thermolysis

Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical, chemical or enzymatic disruption technology. In this study, a novel thermolysis method was used to disrupt E. coli cells to release a recombinant thermostable esterase. We found that heat treatment of E. coli was highly effective to destroy the integrity of bacterial cell walls and release the recombinant hyperthermophilic esterase at temperatures above 60 degrees C. The effects of temperature, pH and cell concentration on the efficiency of cell disruption were examined. The most effective temperature for cell disruption was at 80 degrees C. The pH and cell concentration had only minor effect on the release of the hyperthermophilic esterase. In addition, we found that the hyperthermophilic esterase could be purified at the early stage of the thermolysis, which is a major advantage of the thermolysis method. Finally, a comparison between thermolysis and traditional methods for the disruption of cells and the release of the thermostable enzyme was made.     

Use of a micro-expanded bed containing immobilised lysozyme for cell disruption in flow injection analysis

A method for cell disruption in Flow Injection Analysis (FIA) systems has been developed. The principle involves on-line cell disruption by means of immobilised lysozyme followed by an ultrasonic treatment. In order to avoid flow problems in the analytical system, the lysozyme was immobilised to Streamline^reg that was used in an expanded bed in the flow system. Samples of suspensions of Micrococcus lysodeikticus were treated and the success of the treatment was evaluated in terms of released protein and as a decrease in the optical density at 450 nm. The new technology offers a powerful tool in flow injection analyses for quantification of intracellular compounds. The concept of integration, i.e. combining cell disruption with handling of cell debris and assay procedure in one continuous flow process facilitates its use and increases the probability of reaching reproducible and reliable results.     

Bioenergetics and cytoplasmic membrane stability of the extremely acidophilic, thermophilic archaeon Picrophilus oshimae

Picrophilus oshimae is an extremely acidophilic, thermophilic archaeon that grows optimally at 60°C and at pH 0.7. It is an obligatory acidophile that does not grow at pH values above 4.0. The proton motive force in respiring cells is composed of a large transmembrane pH gradient, inside less acid, and a reversed transmembrane electrical potential, inside positive. Cells maintain an intracellular pH at around 4.6 at extracellular pH values ranging from 0.8 to 4.0. Above pH 4.0 cells lyse rapidly and lose their viability. Liposomes prepared from lipids derived from P. oshimae have an extremely low proton permeability at acidic pH. However, at neutral pH, the lipids are unable to assemble into regular liposomal structures. These observations suggest that the loss of viability and cell integrity above pH 4.0 is due to an impairment of the barrier function of the cytoplasmic membrane. Received: July 18, 1997 / Accepted: November 25, 1997     

Multiplication cellulaire et production lipidique en culture in vitro chez Taphrina insititiae et Taphrina pruni, parasites de Prunus domestica

Cellular Multiplication and Lipid Synthesis during in vitro Culture of Taphrina insititiae and Taphrina pruni, Parasites of Prunus domestica. Two Taphrina species were grown on “yeast extract/glucose” and harvested at regular intervals. Their growth was estimated by dry mycelial weight and cell nitrogen. Lipids were recovered and measured by weighing. Phospholipids, which form the major portion of polar lipids, were measured by phosphorus assay. Lipidic and nitrogenous products were studied with regard to the evolution of the cultures during the phase of active growth. The cells accumulated fats, but during the phase of autolysis, fat content decreased. Production of nitrogenous components was maximal during the autolytic phase. Phospholipids varied as nitrogenous and inversely to neutral lipids. There was apparently an antagonism between nitrogen assimilation and synthesis of neutral lipids. Different functions of neutral and polar lipids may be involved, so that the former act as a reserve material and the latter represent an essential structural cell constituent. Fatty acids were analysed by gas liquid chromatography at various culture periods. Fatty acids from polar as well as those from neutral lipids exhibited great variations with regard to culture evolution. The ratio insaturated/saturated fatty acids is greater in polar lipids than in neutral lipids. These variations are discussed with regard to their consequences for the function of cell membranes.     

pH-Sensitive Liposomes

Abstract

pH sensitive liposomes are lipid compositions that can be destabilized when the external pH is changed; usually from a neutral or slightly alkaline pH to an acidic pH. They are designed to circumvent delivery of liposome contents to the lysosomes of cells following internalization of the vesicle via the endocytic pathway. In the majority of compositions, a lipid containing a pH titratable group is mixed with phosphatidylethanolamine containing unsaturated acyl chains in a molar ratio (pH sensitive component/PE) of 1/4 or greater. There are five major groups of phosphatidylethanolamine containing pH-senstive lipid compositions. These can be classified by their acid-titratable component: phospholipids, acylated amino acids, fatty acids, cholesterol derivatives and miscellaneous double chain amphiphiles. The biophysical mechanism of action involves a transition of the lipids from the lamellar phase to the hexagonal phase. In cell culture, pH sensitive vesicles can increase the delivery of fluorescent markers, proteins, cytotoxic compounds, RNA and DNA into the cytoplasm. The mechanism of delivery is suggested to involve the destabilization of the liposome in the endosome as the pH is reduced from 7.4 to 5.0 and subsequent destabilization of, or fusion with, the endosomal membrane; some of the liposome contents are introduced into the cytoplasm. In most cases, the extent of liposome contents delivery into the cytoplasm is less than 1% of the amount that becomes cell associated. However further studies, with more reliable assays to differentiate cytoplasmic from lysosomal delivery, are required to place an exact value on this efficiency. The efficiency of pH sensitive liposomes in vivo is limited by stability of certain of the liposome compositions in serum and targeting to the appropriate cell. Cholesterol hemisuccinate is a particularly attractive component for in vivo use since it stabilizes the liposome when in serum at pH 7.4. The use of pH sensitive liposomes in drug delivery should continue to expand due to the increasing number of macromolecular therapeutic agents with intracellular targets.     

Measurement of the specific lipid content of attached cells in microtiter cultures

A method has been described to quantify intracellular neutral lipid content in attached cells in microtiter cultures. The procedure was based on oil red O staining of neutral lipid and Coomassie brilliant blue G-250 staining of total cellular protein. Results were expressed as the ratio of lipid to protein, the "specific lipid content" index. This measurement was shown to closely correspond to actual lipid per cell measurements under experimental conditions. The procedure was specific for neutral lipids and sensitive (greater than or equal to 50 ng triglyceride/well). Additionally, cell proliferation measurements could be made simultaneously, using protein staining data. Chromatic endpoints were measured using a spectrophotometer capable of reading individual wells of a microtiter plate. The procedure is recommended for applications in which the endpoint is neutral lipid droplet accumulation in attached cultured cells.     

Alkylated derivatives of poly(ethylacrylic acid) can be inserted into preformed liposomes and trigger pH-dependent intracellular delivery of liposomal contents

Poly(ethylacrylic acid) (PEAA) is a pH-sensitive polymer that undergoes a transition from a hydrophilic to a hydrophobic form as the pH is lowered from neutral to acidic values. In this work we show that pH sensitive liposomes capable of intracellular delivery can be constructed by inserting a lipid derivative of PEAA into preformed large unilamellar vesicles (LUV) using a simple one step incubation procedure. The lipid derivatives of PEAA were synthesized by reacting a small proportion (3%) of the carboxylic groups of PEAA with C₁₀ alkylamines to produce C₁₀-PEAA. Incubation of C₁₀-PEAA with preformed LUV resulted in the association of up to 8% by weight of derivatized polymer with the LUV without inducing aggregation. The resulting C₁₀-PEAA-LUV exhibited pH-dependent fusion and leakage of LUV contents on reduction of the external pH below pH 6.0 as demonstrated by lipid mixing and release of calcein encapsulated in the LUV. In addition, C₁₀-PEAA-LUV exhibited pH dependent intracellular delivery properties following uptake into COS-7 cells with appreciable delivery to the cell cytoplasm as evidenced by the appearance of diffuse intracellular calcein fluorescence. It is demonstrated that the cytoplasmic delivery of calcein by C₁₀-PEAA-LUV could be inhibited by agents (bafilomycin or chloroquine) that inhibit acidification of endosomal compartments, indicating that this intracellular delivery resulted from the pH-dependent destabilization of LUV and endosomal membranes by the PEAA component of the C₁₀-PEAA-LUV. It is concluded that C₁₀-PEAA-LUV represents a promising intracellular delivery system for in vitro and in vivo applications.     

Alteration of the phospho- or neutral lipid content and fatty acid composition in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> due to acid adaptation mechanisms for hydrochloric,acetic and lactic acids at pH 5.5 or benzoic acid at neutral pH

This study provides a first approach to observe the effects on Listeria monocytogenes of cellular exposure to acid stress at low or neutral pH, notably how phospho- or neutral lipids are involved in this mechanism, besides the fatty acid profile alteration. A thorough investigation of the composition of polar and neutral lipids from L. monocytogenes grown at pH 5.5 in presence of hydrochloric, acetic and lactic acids, or at neutral pH 7.3 in presence of benzoic acid, is described relative to cells grown in acid-free medium. The results showed that only low pH values enhance the antimicrobial activity of an acid. We suggest that, irrespective of pH, the acid adaptation response will lead to a similar alteration in fatty acid composition [decreasing the ratio of branched chain/saturated straight fatty acids of total lipids], mainly originating from the neutral lipid class of adapted cultures. Acid adaptation in L. monocytogenes was correlated with a decrease in total lipid phosphorus and, with the exception of cells adapted to benzoic acid, this change in the amount of phosphorus reflected a higher content of the neutral lipid class. Upon acetic or benzoic acid stress the lipid phosphorus proportion was analysed in the main phospholipids present: cardiolipin, phosphatidylglycerol, phosphoaminolipid and phosphatidylinositol. Interestingly only benzoic acid had a dramatic effect on the relative quantities of these four phospholipids.     

Medium pH in submerged cultivation modulates differences in the intracellular protein profile of Fusarium oxysporum

Fusarium oxysporum is a filamentous fungus that damages a wide range of plants and thus causes severe crop losses. In fungal pathogens, the genes and proteins involved in virulence are known to be controlled by environmental pH. Here, we report the influence of culture-medium pH (5, 6, 7, and 8) on the production of degradative enzymes involved in the pathogenesis of F. oxysporum URM 7401 and on the 2D-electrophoresis profile of intracellular proteins in this fungus. F. oxysporum URM 7401 was grown in acidic, neutral, and alkaline culture media in a submerged bioprocess. After 96?hr, the crude extract was processed to enzyme activity assays, while the intracellular proteins were obtained from mycelium and analyzed using 2D electrophoresis and mass spectrometry. We note that the diversity of secreted enzymes was changed quantitatively in different culture-medium pH. Also, the highest accumulated biomass and the intracellular protein profile of F. oxysporum URM 7401 indicate an increase in metabolism in neutral–alkaline conditions. The differential profiles of secreted enzymes and intracellular proteins under the evaluated conditions indicate that the global protein content in F. oxysporum URM 7401 is modulated by extracellular pH.     

Regulation of intracellular pH in bovine oocytes and cleavage stage embryos.

This study investigated the mechanisms for the regulation of intracellular pH in bovine oocytes and embryos. Na(+)/H(+) antiporter activity for the regulation of intracellular pH in the acid to neutral range was detected in both in vitro matured bovine oocytes and in vitro produced embryos. However, the activity of the antiporter was significantly reduced in oocytes compared to 2-cell, 4-cell, and 8-cell embryos. HCO(3)(-)/Cl(-) exchanger activity could be detected in oocytes and embryos using the chloride removal method, however the ability of this transporter to regulate intracellular pH against an alkaline load was poor and intracellular pH could not be re-established. The inability of the HCO(3)(-)/Cl(-) exchanger to adequately regulate intracellular pH was further highlighted by the arrest of embryos at the 8-16 cell stage when challenged with a small alkaline load. Therefore, bovine embryos are extremely sensitive to alterations in intracellular pH above the resting value of around 7.2. This sensitivity could account in part for impaired development and viability of bovine embryos produced in vitro.     

Intracellular pH of Thermoplasma acidophila

Thermoplasma acidophila, a mycoplasma-like organism, was grown at 56 °C and pH 2. The intracellular pH of this organism is close to neutral as measured by the distribution of a radioactive weak organic acid, 5,5-dimethyl-2,4-oxazolidinedione, across the plasma membrane. The cell can maintain the pH gradient when subjected to heat or to metabolic inhibitors. Our experiments seem to indicate that a major portion of the pH gradient is not maintained by active processes, but rather by a Donnan potential across the cell membrane.     

Interaction of maize actin-depolymerising factor with actin and phosphoinositides and its inhibition of plant phospholipase C.

We have previously reported the isolation of three Zea mays genes that encode actin-depolymerising factors/cofilins, a family of low molecular weight actin regulating proteins. In the present study, we have characterised one of these proteins, ZmADF3. We report that ZmADF3 binds G-actin with a 1:1 stoichiometry, and that the interaction with F-actin is pH-sensitive. ZmADF3 co-sediments mainly with F-actin at pH 6.0 and mainly with G-actin at pH 9.0. This response is more similar to that of vertebrate cofilin and ADF than to that of Acanthamoeba actophorin which, although more similar in primary sequence to ZmADF3, is not pH sensitive. However, ZmADF3 requires a more basic environment to depolymerise actin relative to either vertebrate ADF or cofilin. Filaments decorated with ZmADF3 at low pH are very rapidly depolymerised upon raising the pH, which is consistent with a severing mechanism for the disruption of actin filaments. Also, we demonstrate that ZmADF3 binds specific polyphosphatidylinositol lipids, especially phosphatidylinositol 4,5-bisphosphate (PIP₂), and we show that this binding inhibits the actin-depolymerising function of ZmADF3. Moreover, we show that a consequence of ZmADF3 binding PIP₂ is the inhibition of the activity of polyphosphatidylinositol specific plant phospholipase C, indicating the possibility of reciprocal modulation of this major signalling pathway and the actin cytoskeleton.     

Fluorescence and circular dichroism studies on human serum low density lipoprotein particles and lipid-depleted derivatives

The low density liporpotein from human serum, and derivitives prepared free of neutral lipids and total lipids, have been studied by fluorescence and circular dichorism methods. Removal of the neutral lipids had little effect on the tryptophan fluorescence at neutral pH. However, by the criteria of circular dichroism, over the range of 200 nm to 250 nm, there was a reduction in secondary structure of over 75%. Removal of the remaining phospholipids resulted in a qualitatively different structure by both fluorescence and circular dichroism criteria. Neutral lipids were removed from LDL in a step-wise fashion in order to determine the exact amount of neutral lipid required for the native circular dichroism spectrum. The circular dichroism band intensity was constant until approximately 10% of the total cholesterol (as cholesterol ester) remained. The intensity then abruptly dropped as more cholesterol was removed. We concluded that the two spectroscopic methods report on two distinct aspects of LDL structure. The tryptophan fluorescence appears to be sensitive to the presence of phospholipids. The circular dichroism, however, appears to be sensitive to the binding of a small amount of neutral lipid. These findings suggest that a functional and geometric separation of binding sites may exist for these two classes of lipids. Such a distinction is predicted by the icosohedral model of the quaternary structure of LDL. In this model, the phospholipids are located on the surface of the particle, in the holes of an icosohedrally symmetric surface network of protein subunits; the neutral lipids are located in the particle core. Finally, we suggest that functional significance may be attached to our finding that relatively few cholesterol ester molecles are needed to maintain the native secondary structure of LDL. This provides a mechanism whereby the amount of bound neutral lipid could be raised or lowered (for transport and transfer to cells) without affecting the protein in any structurally significant manner.     

Intermediate filaments: Detection of lectin-like activity in purified alcoholic hyaline

Alcoholic hyaline, an intracellular, filamentous (10 nm) aggregate isolated from human alcoholic livers, bound the glycoprotein enzyme, horseradish peroxidase, in a specific and reproducible manner. Using a solid-phase assay system consisting of adsorbed alcoholic hyaline, we have shown that this binding is thermolabile, relatively insensitive to both pH extremes and high ionic strength, and highly sensitive to the presence of neutral and amino sugars. The results suggest that the binding of horseradish peroxidase is not a passive adsorption but rather an “active” phenomenon involving carbohydrate groups on the enzyme. The presence of an intracellular, filament-associated lectin is strongly indicated.     

Ratiometric fluorimetric and colorimetric probe for sensing and imaging pH changes in living cells

Cell, enzyme, and tissue activity in living organisms are closely related to intracellular pH. Detecting the changes of intracellular pH is important to understanding the physiological and pathological changes in the process of crucial cell metabolism. A pH probe (HTBI) based on hemicyanine was synthesized. The probe solution displayed a marked colour change from yellow to amaranth with the pH increase from neutral to basic; simultaneously, the emission spectra showed a significant red shift. The probe exhibited a ratiometric fluorescence emission (F_586nm/F_542nm) characteristic of pK_a 8.82. As expected, HTBI exhibited high sensitivity and selectivity for pH, fine photostability, reversibility, and low cytotoxicity. Therefore, it would be a very useful tool for measuring the intracellular pH changes.     

The effect of ethylenediaminetetra-acetate on Pseudomonas alcaligenes and the composition of the bacterial cell wall

1. EDTA in borate buffer has a marked bactericidal effect on Pseudomonas alcaligenes, which is more sensitive than Pseudomonas aeruginosa. The bactericidal effect is accompanied by solubilization of lipopolysaccharide and release of intracellular solutes. These effects are more pronounced at pH9.2 than 7.1. 2. Cell walls of P. alcaligenes were prepared and from them were obtained the readily extracted lipids and the fractions given by treatment with aqueous phenol. 3. The cell walls and the above components were analysed and results are compared with those for P. aeruginosa. 4. Lipopolysaccharide obtained by treatment of cell walls with aqueous phenol is contaminated with glycosaminopeptide to a variable extent. 5. The lipopolysaccharide contains less neutral sugar but more phosphorus than the lipopolysaccharide of P. aeruginosa; fucosamine is not a component of the lipopolysaccharide of P. alcaligenes.     

Experimental selection of long‐term intracellular mycobacteria

Some intracellular bacteria are known to cause long‐term infections that last decades without compromising the viability of the host. Although of critical importance, the adaptations that intracellular bacteria undergo during this long process of residence in a host cell environment remain obscure. Here, we report a novel experimental approach to study the adaptations of mycobacteria imposed by a long‐term intracellular lifestyle. Selected Mycobacterium bovis BCG through continuous culture in macrophages underwent an adaptation process leading to impaired phenolic glycolipids (PGL) synthesis, improved usage of glucose as a carbon source and accumulation of neutral lipids. These changes correlated with increased survival of mycobacteria in macrophages and mice during re‐infection and also with the specific expression of stress‐ and survival‐related genes. Our findings identify bacterial traits implicated in the establishment of long‐term cellular infections and represent a tool for understanding the physiological states and the environment that bacteria face living in fluctuating intracellular environments.     

A comparative study on effective cell disruption methods for lipid extraction from microalgae

Aims: To compare effective cell disruption methods for lipid extraction from fresh water microalgae. Methods and Results: Chlorella sp., Nostoc sp. and Tolypothrix sp. were isolated from fresh water ponds in and around Gandhigram, Dindigul District, Tamilnadu, India, and used for lipid extraction. Different methods, including autoclaving, bead beating, microwave, sonication and a 10% NaCl solution treatments, were tested to identify the most effective cell disruption method. The total lipids from three microalgal species were extracted using a mixture of chloroform and methanol. Fatty acid composition was detected by gas chromatography (GC). Nostoc sp. and Tolypothrix sp. showed higher oleic acid content of 13·27 mg g^?1 dw and 17·75 mg g^?1 dw, respectively, whereas Chlorella sp. had high linoleic acid content of 17·61 mg g^?1 dw when the cells were disrupted using the sonication method. Conclusions: Finally, the sonication method was found to be the most applicable and efficient method of lipid extraction from microalgae. The highest lipid content was extracted from Chlorella sp. Significance and Impact of the Study: In biodiesel production from microalgae, lipid extraction is a crucial step and important as cell disruption comes in this step. Therefore, the appropriate cell disruption method and device is a key to increase the lipid extraction efficiency.     

Sodium Dependency of the Photosynthetic Electron Transport in the Alkaliphilic Cyanobacterium Arthrospira platensis

Arthrospira (Spirulina) platensis (A. platensis) is a model organism for investigation of adaptation of photosynthetic organisms to extreme environmental conditions: the cell functions in this cyanobacterium are optimized to high pH and high concentration (150–250 mM) of Na⁺. However, the mechanism of the possible fine-tuning of the photosynthetic functions to these extreme conditions and/or the regulation of the cellular environment to optimize the photosynthetic functions is poorly understood. In this work we investigated the effect of Na-ions on different photosynthetic activities: linear electron transport reactions (measured by means of polarography and spectrophotometry), the activity of photosystem II (PS II) (thermoluminescence and chlorophyll a fluorescence induction), and redox turnover of the cytochrome b ₆ f complex (flash photolysis); and measured the changes of the intracellular pH (9-aminoacridine fluorescence). It was found that sodium deprivation of cells in the dark at pH 10 inhibited, within 40 min, all measured photosynthetic reactions, and led to an alkalinization of the intracellular pH, which rose from the physiological value of about 8.3–9.6. These were partially and totally restored by readdition of Na-ions at 2.5–25 mM and about 200 mM, respectively. The intracellular pH and the photosynthetic functions were also sensitive to monensin, an exogenous Na⁺/H⁺ exchanger, which collapses both proton and sodium gradients across the cytoplasmic membrane. These observations explain the strict Na⁺-dependency of the photosynthetic electron transport at high extracellular pH, provide experimental evidence on the alkalization of the intracellular environment, and support the hypothesized role of an Na⁺/H⁺ antiport through the plasma membrane in pH homeostasis (Schlesinger et al. (1996). J. Phycol. 32, 608–613). Further, we show that (i) the specific site of inactivation of the photosynthetic electron transport at alkaline pH is to be found at the water splitting enzyme; (ii) in contrast to earlier reports, the inactivation occurs in the dark and, for short periods, without detectable damage in the photosynthetic apparatus; and (iii) in contrast to high pH, Na⁺ dependency in the neutral pH range is shown not to originate from PSII, but from the acceptor side of PSI. These data permit us to conclude that the intracellular environment rather than the machinery of the photosynthetic electron transport is adjusted to the extreme conditions of high pH and high Na⁺ concentration.