Spectrophotometric method of determining the osmotic resistance of lysosomes isolated from animal tissue

Rapid spectrophotometric determination of animal tissue lysosome osmoresistance is described. The duration of analysis is decreased by 5-6 h, as compared to enzymatic method. The process of lysosome degradation was linear at 0.7 M sucrose concentration. The dependence of sucrose molarity on the extent of lysosome lysis was complex. The lysosome lysis rate rapidly increased at sucrose molarity less than 0.7 M. Lysosome incubation at 0.4 degrees C for 24 h increased their osmotic sensitivity within the whole investigated osmotic pressure range. Isolated lysosome osmoresistance may be used as an index of dynamic structural and functional state of these particles and the extent of their degradation caused by various physical and chemical factors in vivo and in vitro. The measure of the initial absorption (A520) and the initial lysosome lysis rate (delta A520/min), as well as biological half-life may be the index of such an effect.     

A rapid spectrophotometric method of determining heat-resistant lysosomes in animal tissues

Rapid spectrophotometric method for the determination of thermoresistance in tissue animal lysosomes is described. The of analysis is decreased by 5-6 h, in comparison with enzymatic technique. The determination regimen was chosen in such a way that the process of lysosomal lysis was linear. The dependence of the incubation mixture temperature on the degree of lysosomal lysis was complex. The rate of lysosomal lysis rapidly increased at greater than 37 degrees C. Lysosome incubation at 0-4 degrees C for 24 h decreased its hypothermal (t = 10-30 degrees C), but not hyperthermal (t greater than 37 degrees C) sensitivity. Isolated lysosome thermoresistance may be used as an index of its stability and labialization in vivo and in vitro by various physico-chemical factors. The percentage of initial absorption (A520) and the initial rate of lysosomal lysis (delta A520/min), as well as melting temperature (Tmel) and biological half-life (t1/2) may be the measurements of such effect.     

Enrichment of sulfate-reducing bacteria and resulting mineral formation in media mimicking pore water metal ion concentrations and pH conditions of acidic pit lakes

Acid mine drainage sites are extreme environments with high acidity and metal ion concentrations. Under anoxic conditions, microbial sulfate reduction may trigger the formation of secondary minerals as a result of H2S production and pH increase. This process was studied in batch experiments with enrichment cultures from acidic sediments of a pit lake using growth media set at different pH values and containing elevated concentrations of Fe2? and Al3?. At initial pH values of 5 and 6, sulfate reduction occurred shortly after inoculation. Sulfate- reducing bacteria affiliated to the genus Desulfosporosinus predominated the microbial communities as shown by 16S rRNA gene analysis performed at the end of the incubation. At initial pH values of 3 and 4, sulfate reduction and cell growth occurred only after an extended lag phase, however, at a higher rate than in the less acidic assays. At the end of the growth phase, enrichments were dominated by Thermodesulfobium spp. suggesting that these sulfate reducers were better adapted to acidic conditions. Iron sulfides in the bulk phase were common in all assays, but specific aluminum precipitates formed in close association with cell surfaces and may function as a detoxification mechanism of dissolved Al species at low pH.     

Dependence of erythromycin biosynthesis on the active acidity of the medium]

Dependence of erythromycin biosynthesis on the medium active acidity was studied by the following methods: by changing pH of the initial medium, by changing the concentration of the medium components determining the active acidity of the culture, by using buffer mixtures by automatic control of pH. It was found that pH of the initial medium within 5.7-8.1 had no effect on the culture growth. Biosynthesis of erythromycin markedly decreased at pH 6.3 or lower. The values of pH within 6.6-7.5 (optimal values 6.7-6.9) were favourable for the antibiotic biosynthesis. At pH 6.2-6.3 the antibiotic accumulation was equal to 5-10 per cent of the control.     

Self-association and stability of the ApoE isoforms at low pH: implications for ApoE-lipid interactions

Apolipoprotein E (apoE) isoforms are known to differentially accumulate in the lysosomes of neuronal cells, and the deleterious effects of the apoE4 isoform in Alzheimer's disease may relate to its properties at the low lysosomal pH. However, the effect of pH on the molecular properties of full-length apoE is unclear. Here we examine the pH dependence of the monomer-dimer-tetramer reaction, of lipid binding, and of the stability of the three major apoE isoforms. Using FRET measurements, we find that the association-dissociation behavior of apoE proteins changes dramatically with changes in pH. At pH 4.5, approximating the pH of the lysosome, rate constants for association and dissociation are 2-10 times faster than those at pH 7.4. Aggregation beyond the tetrameric form is also more evident at lower pH values. Stability, as measured by urea denaturation at pH 4.5, is found to be considerably greater than that at neutral pH and to be isoform dependent. Lipid binding, as measured by turbidity clearance of unilamellar vesicles of DMPC, is faster at acidic pH values and consistent with our previous hypothesis that it is only the monomeric form of apoE that binds lipid tightly. Since apoE is more stable at pH 4.5 than at neutral pH, the more rapid apoE-lipid interactions at low pH are not correlated with the stability of the apoE isoforms, but rather to the faster association-dissociation behavior. Our results indicate that pathological behavior of apoE4 may arise from altered molecular properties of this protein at the acidic pH of the lysosome.     

Low pH as an inhibiting factor in the transition from mesophilic to thermophilic phase in composting

During composting of household waste, the acidity of the material affects the process during the initial phase of rising temperature. In this study, the effects of temperature (36-46 degrees C) and pH (4.6-9.2) on the respiration rate during the early phase of composting were investigated in two different composts. A respiration method where small compost samples were incubated at constant temperature was used. The respiration rate was strongly reduced at 46 degrees C and pH below 6, compared to composts with a higher pH or lower temperature. The combination of high temperature and low pH is a possible adverse factor in large-scale composting of food waste.     

低pH条件对紫花苜蓿结瘤的影响及机制初探

探讨了低pH条件下紫花苜蓿根毛变形和结瘤受到的影响及其机制。结果表明,在低pH条件下,初生根伸长和根瘤菌OD600值显著下降,根共生结瘤受到明显抑制。在接种根瘤菌、不加NF的条件下,pH5.0、pH4.7、pH4.5、pH4.2处理的根毛变形率分别比对照(pH6.5)减少了44.1%、56.4%、60.0%和69.0%;在加入NF、不接种根瘤菌的情况下,低pH(4.5)处理,根毛的变形也比对照(pH6.5)减少了45.9%。结果暗示,低pH条件下苜蓿结瘤初期的结瘤信号传导受阻,这可能是导致酸性条件下苜蓿结瘤减少的重要原因之一。     

Microflora of the aerobic preservation of directly brined green olives from Hojiblanca cultivar

New procedures for the preservation stage of ripe olives from Hojiblanca cultivar were studied. An aerobic fermentative process was used with initial pH correction (0.3% acetic acid) and various NaCl concentrations: 6, 3 and 0% (w/v) in tap water. Treatments were carried out at industrial level and the spontaneous changes monitored. At initial salt concentrations of 6 and 3% (w/v) NaCl, pH rose progressively, reaching 4.3 at equilibrium maintaining during this period a constant free lactic acidity of around 0.4% (w/v). When the initial solution was tap waste, however, the pH decreased rapidly to stabilize at about 3.7, and lactic acidity increased continuously to reach values over 1% (w/v) at the end of the preservation process. In all treatments aeration effectively purged the carbon dioxide from the preservation brines, preventing shrivelling of olives. The microbial growth was strongly influenced by the initial NaCl concentration. At 6 and 3%, only yeasts grew, the most abundant being Pichia membranaefaciens, P. vini, P. fermentans and Hansenula polymorpha. However, when there was no NaCl, lactic acid bacteria colonized the solution. Lactobacillus plantarum and Pediococcus inopinatus were the only species found. In this case there was a co-existence between yeasts and lactic acid bacteria. As the treatment that supported lactic acid bacteria achieved the best final pH and acidity for olive stability, it may help to overcome the obstacles to a lactic fermentative process during the preservation stage of ripe olives from the Hojiblanca cultivar.     

以纸为碳源去除地下水硝酸盐的研究

研究了以纸为碳源和反应介质的生物反应器对水中硝酸盐的去除。结果表明,以纸为碳源的反应器启动快．反硝化反应受温度及水力停留时间影响大。25℃的反硝化速率是14℃的1．7倍。在室温25±1℃,进水硝酸盐氮浓度为45．2mg·L^-1、水力停留时间8．6h时,反应器对硝酸盐氮的去除率在99．6％以上,当水力停留时间为7．2h,氮去除率只有50％。反硝化反应受pH值和溶解氧的影响小,反应进行过程中,纸表面形成了生物膜,纸也被消耗了．采用反应器出水再经活性炭吸附的工艺流程处理高硝酸盐氮地下水,<33．9mg·L^-1的硝酸盐氮完全去除,没有出现NC2-N,最终出水水质DOC<11mg·L^-1。     

The comparative influence of substituted phenols (especially chlorophenols) on yeast cells assayed by electro-rotation and other methods

The toxicity of 31 phenols was studied by electro-rotation of yeast cells. Control yeast cells show both anti-field and co-field rotation, depending upon the field frequency applied. After treatment with supra-threshold amounts of phenols the anti-field rotation is weakened or abolished and a stronger co-field rotation can be seen. The proportion of cells showing the co-field rotation was found to be a sensitive measure of toxicity. Doses of 2.2 mumol/l of pentachlorophenol, or of 0.3 mumol/l of pentabromophenol were detectable after 3 h incubation at pH 4.0. At a given pH, the toxicity of the chlorophenols correlated extremely well with their octanol:water partition coefficients (Pow). The complete set of phenols showed fair overall correlation with Pow, but less good correlation with their acidity constants (pKa). In particular the toxicity of a given phenol was less than predicted from its pKa if the incubation pH was higher than the pKa. Biochemical assays on 23 of the phenols showed that the rotational sensitivity runs closely parallel to the sensitivities of cell growth rate and of the plasmamembrane ATPase, but less closely to the inhibition of purine incorporation. It appears that the electro-rotation method provides a useful and rapid test for the presence of organic ecotoxins. The test enables us to distinguish differences between single cells, and is comparable in sensitivity to biochemical tests that use vesicles or homogenates derived from a cell population.     

Deacylation of penicilloylated penicillin-binding proteins from Micrococcus luteus: kinetic study of thiol-promoted release of the bound penicilloyl moiety

The stability of covalent complexes obtained by labelling penicillin-binding proteins 1-6 from Micrococcus luteus with a radioactive derivative of ampicillin has been examined in the presence of thiols. When the incubation medium contained only 1 mM 2-mercaptoethanol, the complexes were almost unaffected for at least 1 h. If 20 mM dithiothreitol was also included in the medium, the amount of bound radioactivity decreased throughout the incubation period. The breakdown of the complexes derived from penicillin-binding proteins 4 and 5 proceeded very slowly, following an apparent first-order kinetics, whereas the kinetics of deacylation of other penicillin-binding proteins exhibited a biphasic pattern with an initial fast phase followed by a slow one, each of which could be approximated by an apparent first-order reaction. This behavior is explained adequately by a two-step mechanism: the penicilloylated penicillin-binding proteins are first deacylated in a reversible exchange with the added thiol, giving rise to an intermediate thioester; once formed, this intermediate is hydrolysed irreversibly. A simple graphical method has been devised to deduce rate constants from the time course of the reaction. Theoretical curves have been constructed, and they fit experimental data satisfactorily. The results point out that added thiols may effectively interfere with the quantitation of penicillin-binding proteins; therefore, the stability of penicilloylated penicillin-binding proteins should be checked carefully when these protecting agents are included in membrane extracts or incubation media.     

Synthesis and lysis of formate by immobilized cells of Escherichia coli

Formate hydrogenlyase (FHL) activity was induced in a strain of Escherichia coli S13 during anaerobic growth in yeast extract-tryptone medium containing 100 mM formate. The cells obtained at the optimum growth phase were immobilized in 2.5% (w/v) agar gel when 50-60% of the whole cell FHL activity was retained. The immobilized FHL system had good storage stability and recycling efficiency. In the lysis of formate, an increase of formate concentration to 1.18M increased QH(2) (initial) value of the immobilized cell, and subsequently cells, hydrogen evolution, in general, ceased after 6 to 8 of incubation, resulting in incomplete lysis of formate. Presence of small amount of glucose (28 mM) was more or less quantitatively lysed with concomitant disappearence of glucose from the medium. Synthesis of formate from hydrogen and bicarbonate solution by the immobilized cells was also characterized. Presence of glucose (10 mM) in 50 mM bicarbonate solution stimulated formate synthesis by immobilized cells. The pH optimum range, K(m), and specific activity of the immobilized cells for the lysis of formate were 6.8-7.2 0.4M, and 66 mL/g cell-h, respectively. The cells could fix hydrogen to the extent of 24.4% (w/w) of its own wet cell mass in a 72-h reaction cycle. Potentiality of the immobilized FHL system for biotechnological exploitation was discussed.     

High nisin Z production by Lactococcus lactis UL719 in whey permeate with aeration

The influence of controlled pH (5.0–6.5) and initial dissolved oxygen level (0–90% air saturation) on nisin Z production in a yeast extract/Tween 80-supplemented whey permeate (SWP) was examined during batch fermentations with citrate positive Lactococcus lactis subsp. lactis UL719. The total activity corresponding to the sum of soluble and cell-bound activities, as measured by a critical dilution method, was more than 50% lower at pH 5.0 than in the range 5.5–6.5, although the specific production decreased as pH increased. A maximum nisin Z activity of 8200 AU/ml (4100IU/ml) was observed in the supernatant after 8h of culture for pH ranging from 5.5 to 6.5. Prolonging the culture beyond 12h decreased this activity at pH 6.0 and 6.5 but not at pH 5.5 or 5.0. A corresponding increase in cell-bound activity was probably due to adsorption of soluble bacteriocin to the cell wall. Aeration increased cell-bound and total activity to maximum values of 32800 and 41000 AU/ml (16400 and 20500IU/ml), respectively, with an initial level of 60% air saturation after 24h of incubation at pH 6.0. The specific production at 60% or 90% initial air saturation was eight-fold higher than at 0%.     

Overnight lysis improves the efficiency of detection of DNA damage in the alkaline comet assay

The ability to detect DNA damage using the alkaline comet assay depends on pH, lysis time and temperature during lysis. However, it is not known whether different lysis conditions identify different types of DNA damage or simply measure the same damage with different efficiencies. Results support the latter interpretation for radiation, but not for the alkylating agent MNNG. For X-ray-induced damage, cells showed the same amount of damage, regardless of lysis pH (12.3 compared to >13). However, increasing the duration of lysis at 5 degrees C from 1 h to more than 6 h increased the amount of DNA damage detected by almost twofold. Another twofold increase in apparent damage was observed by conducting lysis at room temperature (22 degrees C) for 6 h, but at the expense of a higher background level of DNA damage. The oxygen enhancement ratio and the rate of rejoining of single-strand breaks after irradiation were similar regardless of pH and lysis time, consistent with more efficient detection of strand breaks rather than detection of damage to the DNA bases. Conversely, after MNNG treatment, DNA damage was dependent on both lysis time and pH. With the higher-pH lysis, there was a reduction in the ratio of oxidative base damage to strand breaks as revealed using treatment with endonuclease III and formamidopyrimidine glycosylase. Therefore, our current results support the hypothesis that the increased sensitivity of longer lysis at higher pH for detecting radiation-induced DNA damage is due primarily to an increase in efficiency for detecting strand breaks, probably by allowing more time for DNA unwinding and diffusion before electrophoresis.     

Various properties of immobilized terminal deoxynucleotidyl transferase from the cattle thymus

The effects of temperature, pH, and concentration of sodium cacodylate buffer on the activity of partially purified terminal deoxynucleotidyl transferase from cattle thymus immobilized on BrCN-Sepharose were studied. The enzyme retained at least 60% of the initial activity after 6 h of incubation at 30 degrees in 50 mM potassium phosphate buffer, pH 7.2 in the absence of substrate. Short-term activation of the enzyme during incubation was noticed. The maximum activity of the immobilized preparations was observed in 240-280 mM sodium cacodylate buffer in the reaction mixture, pH 7.5-7.9 at 37-40 degrees.     

Chitosanases in the autolysis of Mucor rouxii

The mycelium of Mucor rouxii reached a 50% degree of lysis after 50 days incubation, and was then stable with the incubation time. The pH of the medium was 4.3 when autolysis began, rising to pH 7.6 after 6 days of autolysis and remaining there for the duration of the experiment. Maximum degradation of mycelium occurs during the first days of autolysis. Glucosamine is present in the culture liquid during all the autolytic process. Enzymes implicated in the degradation of chitosan and chitin were studied in the culture fluid during autolysis. An exochitosanase activity was detected after a day of autolysis, and its activity increased during 20 days of autolysis and afterwards remained constant until the end of the process. An endochitosanase activity was detected in the culture fluid from the beginning of the autolysis, having its maximum activity after 34 days of incubation. Both activities show an optimum pH of 5.5, but the pH range of activity for endochitosanase was broader than for exochitosanase. Both activities were not inhibited by 0.5 mM glucosamine. Activities of the enzymes B-N-acetylglucosaminidase and chitinase were not found. The chitosan content in the cell walls decreased with the incubation time. In these cell walls the chitin content experienced an increase at the beginning of the autolysis, decreasing afterwards. The enzymatic complex obtained from autolyzed cultures of M. rouxii hydrolyzed 2-day-old cell walls of this fungus. The hydrolysis was 21% after 24 h of incubation, liberating glucose and glucosamine. As a consequence protoplasts from M. rouxii germinated spores were obtained with its own lytic enzymes in adequate osmotic conditions. The involvement of chitosanases in the autolysis of this fungus have been studied.     

The influence of substrate concentrations on the rate of insect juvenile hormone biosynthesis by corpora allata of the desert locust in vitro

The rate at which isolated corpora allata of adult female Schistocerca gregaria incorporate [(3)H]farnesenic acid and [(14)C]methionine into C(16)juvenile hormone in vitro was examined at different concentrations of farnesenic acid, methionine, O(2) and H(+) ions. Maximum juvenile hormone biosynthesis is obtained at a farnesenic acid concentration of 20mum. The range of optimum l-methionine concentrations (0.1-0.4mm) encompasses the physiological concentration of this substrate in the haemolymph. Hormone biosynthesis is dependent on O(2), but is not stimulated by hyperbaric oxygen tension. The glands had a maximum synthetic activity at pH8.0, but their activity was more reproducible in the the physiological range pH7.0-7.5. At pH6.5 and less, the synthetic ability was considerably decreased. The relative incorporations of the labelled substrates into methyl farnesoate and C(16) juvenile hormone indicate that [(3)H]farnesenic acid comes into isotopic equilibrium within the gland more rapidly than [(14)C]methionine. The incorporations into methyl farnesoate become stoicheiometric after 20min incubation and into C(16) juvenile hormone after a further 10min. Labelled juvenile hormone is detectable after 10min incubation and the rate of incorporation is constant for up to 4h. It is proposed that the described method may be usefully employed to assess the physiological changes in the enzymic competence of the glands to effect the last two stages in C(16) juvenile hormone biosynthesis.     

The influence of pH on the growth rate and viability of neutrophilic and acidophilic streptomycetes.

The influence of pH on the specific growth rates of two acidophilic and two neutrophilic soil streptomycetes was studied. The acidophiles had maximum growth rates over a broad range from pH 4.5 to 5.5, while the neutrophiles had clearly defined optima at pH 7.0. Mycelium of neutrophiles was less tolerant of acidity than that of acidophiles; both showed decreased viability at pH levels below those which allowed growth. Spores of neutrophiles and acidophiles were equally tolerant of acidity and this may allow the former to survive in acid soils. Both spores and mycelium of acidophiles remained viable at pH levels above those allowing growth.     

Preparation of cross-linked lipase-coated micro-crystals for biodiesel production from waste cooking oil

A dual modification procedure composed of cross-linking and protein coating with K₂SO₄ was employed to modify Geotrichum sp. lipase for catalyzing biodiesel production from waste cooking oil. Compared to single modification of protein coating with K₂SO₄, the dual modification of cross-linking and lipase coating improved catalytic properties in terms of thermostable stability, organic solvent tolerance, pH stability and operational stability in biodiesel production process, although biodiesel yield and initial reaction rate for CLPCMCs were not improved. After five successive batch reactions, CLPCMCs could still maintain 80% of relative biodiesel yield. CLPCMCs retained 64% of relative biodiesel yield after incubation in a pH range of 4-6 for 4 h, and 85% of relative biodiesel yield after incubation in a range of 45-50 °C for 4 h. CLPCMCs still maintained 83% of relative biodiesel yield after both treated in polar organic solvent and non-polar organic solvent for 4 h.     

Influence of acidity and initial substrate temperature on germination of Rhizopus oligosporus sporangiospores during tempe manufacture

J.C. DE REU, F.M. ROMBOUTS AND M.J.R. NOUT. 1995. During the soaking of soya beans according to an accelerated acidification method organic acids were formed, resulting in a pH decrease from 6·0 to 3·9. After 24 h of fermentation at 30°C, lactic acid was the major organic acid (2·1% w/v soak water), while acetic acid (0·3% w/v soak water) and citric acid (0·5% w/v soak water) were also found. During cooking with fresh water (ratio raw beans: water, 1: 6·5) the concentrations of lactate/lactic acid and acetate/acetic acid in the beans were reduced by 45% and 51%, respectively.
The effect of organic acids on the germination of Rhizopus olgosporus sporangiospores was studied in liquid media and on soya beans. Germination in aqueous suspensions was delayed by acetic acid: within 6 h no germination occurred at concentrations higher than 0·05% (w/v incubation medium), at pH 4·0. When soya beans were soaked in the presence of acetic acid, the inhibitory concentration depended on the pH after soaking. Lactic acid and citric acid enhanced germination in liquid medium, but not in tempe.
Inoculation of soya beans with R. oligosporus at various temperatures followed by incubation at 30°C resulted in both increased and decreased periods for the lag phase of fungal growth. A maximum difference of 3 h lag phase was found between initial bean temperatures of 25 and 37°C.
When pure cultures of homofermentative lactic acid bacteria were used in the initial soaking process, less lactic acid and acetic acid was formed during soaking than when the accelerated acidification method was used. This resulted in a reduction of the lag phase before growth of R. oligosporus by up to 4·7 h.