Cyanide-initiated oxygen consumption in autoclaved culture medium containing sugars

The consumption of oxygen initiated by KCN in an autoclaved sugar-containing rinse medium with protoplasts is described. The effect of autoclaving on several sugars was examined. Fructose solutions, followed in decreasing order by glucose, sucrose and sorbitol, were found to contain the largest amount of degraded products that could react with oxygen in the presence of KCN. Mannitol was found to be stable under the autoclaving conditions used in this investigation. KCN generally has an inhibitory effect on respiration, but in some plant tissues, respiration is stimulated by it. Under certain circumstances the degradation artefact described here may confuse interpretation of the results of respiration measurements. The use of autoclaved media containing sugars should be avoided in respiration studies that involve the application of KCN.Abbreviations SHAM salicylhydroxamic acid     

Respiration and metabolic processes of bacteria in autoclaved and gamma-irradiated soil

Samples of chernozem and brown soils were irradiated with gamma rays using doses of 1.5 to 4.5 Mrad, or fractionally autoclaved at 1 and 2 kp/cm². Consumption of oxygen by cellular suspensions of bacteria added to suspensions of sterilized soils was higher than in untreated samples. The increased oxygen consumption indicated an increase in the quantity of a biologically oxidizable substrate which could be released during the irradiation or autoclaving of soils. The amount of oxygen consumed was proportional to the radiation dose or autoclaving time and the pressure used, and was dependent also on the type of soil. The accessible substrate could be immediately, without a lag phase, oxidized by the added microorganism. The extent and rate of oxygen consumption in the sterilized soil samples varied in different microorganisms. It was observed that decomposition of vanillie acid by a cell suspension ofCellulomonas sp. was stimulated in the soil sterilized by radiation. The significance of these findings for the soil metabolic studies is discussed.     

Further studies on autoclaved-induced toxicity in tissue culture media: gauging sugar breakdown by spectrophotometry

Fructose and sucrose were used to investigate cyanide-induced absorption changes after high temperature treatment. By comparing the time-resolved absorbance difference spectra obtained under aerobic conditions with those under aerobic conditions, absorbance changes that are associated with the process of oxygen consumption were identified. The rates of absorbance changes of autoclaved sucrose solution or of autoclaved MS medium (Murashige and Skoog 1962. Physiol. Plant. 15: 473–497) were correlated with those of oxygen consumption measured by polarography and used for determining the toxicity of culture media (Hsiao and Bornman 1991. Physiol. Plant. 81: 55–58). When autoclaved together with sucrose, FeNa-EDTA promotes its degradation. Absorbance change, therefore, is a convenient parameter for measuring not only the extent of carbohydrate breakdown at high temperature but also the relative toxicity of culture media autoclaved under different conditions.     

Sugar degradation during autoclaving: Effects of duration and solution volume on breakdown of glucose

As the autoclaving of a sugar together with other nutrient components enhances its degradation with the associated formation of toxic products, it is advisable to autoclave it separately from other medium components. In such cases, and to prepare growth media of consistent quality, it may also be necessary to adjust the duration of autoclaving according to the volume of the sugar solution. The results of an attempt to fine-adjust the autoclaving procedure are presented.
Glucose, used as model sugar in this investigation, was autoclaved for 20,32 or 44 min in volumes of 0.25. 1.2 and 5 1. The rise in temperature of the solutions was monitored by thermocouples. Glucose degradation was estimated by the rate of cyanide-initiated oxygen consumption. The rates were found to be inversely proportional to the volumes in a semilogarithmic plot. The T-t (temperature-time) value, the area under the solution temperature vs time curve, was found to be an independent variable from which the extent of glucose degradation could be deduced. For a chosen level of thermodegradation of glucose, the duration of autoclaving for solutions of different volumes could be determined graphically from a T-t value vs time plot.     

Studies on cartilage formation. XXII. Investigations of certain oxidative metabolic processes in regenerating articular cartilage

The distal articular surface of the femur was surgically removed in 57 dogs. Succinate dehydrogenase and cytochrome oxidase activities were assayed on postoperative days 7, 20, 26, 33 and 70 in the regenerating, chondrifying articular surface and in the granulation tissue adhering to the capsule. In the 70-day samples, the cyanide-induced inhibition of oxygen consumption was determined and enzyme histochemical reactions (cytochrome oxidase, monoamine oxidase, xanthine oxidase, peroxidase and "catalase") were performed. The succinate dehydrogenase activity was the highest in the early postoperative stage in both tissues. This was followed by a definite decrease and a subsequent significant increase in activity when chondrification took place. Measurement of cytochrome oxidase activity could not reveal any convincing result, presumably because of the properties of the tissues studied. The oxygen consumption by the chondrifying articular surface at 70 days was inhibited to about 50% by cyanide, and about 90% inhibition was observed in the tissue adhering to the capsule. The cells of the regenerating articular surface possess cytochrome oxidase and a cyanide- (and sodium azide-) resistant oxidase activity. The enzyme activity of the cartilaginous islets exceeded that of their connective tissue environment. The cytochrome oxidase activity increased in the cells during cartilage differentiation. Presumably, some further cyanide-sensitive and cyanide-resistant oxidases are present in chondroblasts and young chondrocytes.     

Amperometric determination of total assimilable sugars in fermentation broths with use of immobilized whole cells

A microbial sensor consisting of immobilized living whole cells of Brevibacterium lactofermentum and an oxygen electrode was prepared for continuous determination of total assimilable sugars (glucose, fructose and sucrose) in a fermentation broth for glutamic acid production. Total assimilable sugars were evaluated from oxygen consumption by the immobilized microorganisms. When a sample solution containing glucose was applied to the sensor system, increased consumption of oxygen by the microorganisms caused a decrease in the dissolved oxygen around the Teflon membrane of the oxygen electrode and the current of the electrode decreased markedly with time until steady state was reached. The response time was ≈ 10 min by the steady state method and 1 min by the pulse method. A linear relationship was found between the decrease in current and the concentration of glucose (<1 mM), fructose (<1 mM) and sucrose (<0.8 mM). The ratio of the sensitivity of the microbial sensor to glucose, fructose and sucrose was 1.00:0.80:0.92. The decrease in current was reproducible to within 2% of the relative standard deviation when a sample solution containing glucose (0.8 mM) was employed for experiments. The selectivity of the microbial sensor for assimilable sugars was satisfactory for use in the fermentation process. The additivity of the response of the microbial sensor for glucose, fructose and sucrose was examined. The difference between the observed and calculated values was within 8%. The microbial sensor was applied to a fermentation broth for glutamic acid production. Total assimilable sugars can be determined by the microbial sensor which can be used for more than 10 days and 960 assays.     

The coupling coefficient as an index of junctional conductance

Summary Toad bladder epithelial cells were isolated under mild conditions in a calcium-free medium; they were found to exclude trypan blue, to consume oxygen, and to respond to vasopressin with an increased rate of oxygen consumption. Since isolated toad bladder epithelial cells are mostly spherical in shape, the cell diameter can be accurately measured with an ocular micrometer of an inverted microscope. Epithelial cells swelled by 29±3% in the presence of KCN. This cyanide-induced swelling of cells was prevented by amiloride or, alternatively, by replacing NaCl by equiosmotic amounts of mannitol in the Ringer's fluid. Cells incubated in the presence of vasopressin swelled by 10±2%. Vasopressin and KCN acted synergistically in enhancing cell volume. Ouabain caused cells to swell by 9±2%, and this effect was not additive to the swelling seen with vasopressin. These observations are in accord with the theory of Leaf and his associates, that the predominant effect of vasopressin is to enhance sodium entry into the transporting epithelial cells of the toad urinary bladder.     

Effects of autoclave-induced carbohydrate hydrolysis on the growth ofBeta vulgaris cells in suspension

Media of de Greef & Jacobs (1979) were autoclaved either with all the nutrient components in a single vessel (medium 1) or with the following components in separate vessels: FeNa–EDTA+CaCl₂ (medium 2), FeNa–EDTA+NaH₂PO₄ (medium 3) or sucrose (medium 4). Medium 5 was prepared by autoclaving FeNa–EDTA+NaH₂PO₄ and sucrose in two separate vessels. It was found that the dry mass yield of cell suspensions ofBeta vulgaris was lowest in medium 1, followed by media 2 and 3. There was no significant difference among media 3, 4, and 5.The plot of dry mass yield of the cell suspensions against the rates of cyanide-initiated oxygen consumption which indicate the extent of carbohydrate hydrolysis of the media during autoclaving, indicated the presence of a threshold rate of about 17–20 nmol ml^–1 min^–1. Dry mass yield of the suspensions decreased rapidly when the rate exceeded this value.For media with glucose as the source of carbohydrate, the rate of cyanide-initiated oxygen consumption exceeded the threshold value by a factor of 1.5 to 2, depending on the volume of the media autoclaved.Abbreviations FeNa-EDTA ferric monosodium ethylenediamine-tetraacetic acid     

Studies on Amino Acid Contents of Processed Soybean

The influence of sugars on the heat destruction of the basic and sulphur containing amino acids of soybean products, soybean protein and also pure amino acids has been investigated.

Amino acids were estimated by microbiological assay procedure. Cystine was also determined chromatographically.

Lysine, arginine and histidine were destroyed in different ways behaved when soybean products, soybean protein or pure amino acids mixed with and without sugars were autoclaved; and the condition of added sugars tended to influence the way of destruction.

Cystine was the most heat-labile amino acid. But the destruction did not appear to be ascribed to added sugars except 50% ethyl alcohol-soluble sugar solution of defatted soybean flour. This finding was also substantiated by the chromatographic analysis.

Methionine, in soybean products and soybean protein or as pure amino acid with and without the addition of sugars, was not influenced by autoclaving.     

ISOLATION OF EXINES FROM GYMNOSPERM POLLEN

Exines of certain Gymnosperms spontaneously separate from the intine during the process of hydration preceding pollen germination. Exines of pollen of Calocedrus decurrens, Chamaecyparis lawsoniana, Juniperus occidentalis, Sequoia sempervirens, and Pseudotsuga menziesii were isolated from hydrated, autoclaved pollen. Free exines were purified by centrifugation on a discontinuous sucrose gradient of densities 1.14 to 1.27 g/ml. The outer intine dissolved on autoclaving. This method may be applicable to a wide range of genera. Purified exines are of potential use in chemical analyses of sporopollenin and in production of antibodies to exine.     

Hive‐stored pollen of honey bees: many lines of evidence are consistent with pollen preservation,not nutrient conversion

Honey bee hives are filled with stored pollen, honey, plant resins and wax, all antimicrobial to differing degrees. Stored pollen is the nutritionally rich currency used for colony growth and consists of 40–50% simple sugars. Many studies speculate that prior to consumption by bees, stored pollen undergoes long‐term nutrient conversion, becoming more nutritious ‘bee bread’ as microbes predigest the pollen. We quantified both structural and functional aspects associated with this hypothesis using behavioural assays, bacterial plate counts, microscopy and 454 amplicon sequencing of the 16S rRNA gene from both newly collected and hive‐stored pollen. We found that bees preferentially consume fresh pollen stored for <3 days. Newly collected pollen contained few bacteria, values which decreased significantly as pollen were stored >96 h. The estimated microbe to pollen grain surface area ratio was 1:1 000 000 indicating a negligible effect of microbial metabolism on hive‐stored pollen. Consistent with these findings, hive‐stored pollen grains did not appear compromised according to microscopy. Based on year round 454 amplicon sequencing, bacterial communities of newly collected and hive‐stored pollen did not differ, indicating the lack of an emergent microbial community co‐evolved to digest stored pollen. In accord with previous culturing and 16S cloning, acid resistant and osmotolerant bacteria like Lactobacillus kunkeei were found in greatest abundance in stored pollen, consistent with the harsh character of this microenvironment. We conclude that stored pollen is not evolved for microbially mediated nutrient conversion, but is a preservative environment due primarily to added honey, nectar, bee secretions and properties of pollen itself.     

Improvement of Carob Pods as Feed by Solid-substrate Fermentation

Chopped carob pods were autoclaved in an ammonium salt solution. The solution was absorbed by the pods during autoclaving. The material was inoculated with either Rhizopus oligosporus or Monascus ruber and incubated for 3 d at 30 °C. The growing mycelium penetrated the particles to form a cake-like structure containing ca. 7% protein. The fermentation product contained 73–83% of the original carob sugars, whereas the amount of tannins was considerably lower than in the pods.     

Exogenous sugars overcome incompatibility in hazelnut (Corylus avellana L.)

Summary Petreatment of hazelnut (Corylus avellana L.) stigmas with L M or 2 M sugar solutions before pollination with incompatible pollen, or pollination with 11 mixtures of incompatible pollen and finely ground sugars, prevented rejection of incompatible pollen by the stigma surface. Lower sugar concentrations or lower ratios of sugar to pollen were less effective. No specificity for overcoming incompatibility was observed among 18 simple sugars and related sugar compounds.Oregon Agricultural Experiment Station Technical Paper No. 7613     

Icreased drug permeability in Chinese hamster ovary cells in the presence of cyanide

In this report we investigated whether the modulation of drug permeability in Chinese hamster ovary (CHO) cells was an energy-dependent process. We observed that (1) in the absence of glucose, metabolic inhibitors such as cyanide, azide, and dinitrophenol stimulated the uptake of [³H]colchicine and other drug; (2) cyanide-induced stimulation of drug uptake could be prevented by the presence of metabolizable sugars such as glucose and ribose; (3) cyanide-treated cells were fully viable; (4) on the addition of cyanide and glucose the kinetics of drug permeability changes were very rapid. These data are consistent with the hypothesis that an energy-dependent membrane barrier against the uptake of a variety of drugs was operative in CHO cells.The nature of this energy-dependent membrane barrier was examined in colchicine-resistant mutants (CH^RC4 and CH^RC5 cells) previously characterized as membrane mutants with greatly reduced drug permeability (Ling and Thompson, (1974) J. Cell Physiol. 83, 103–116). The mutants were more refractile to the cyanide-induced stimulation of drug permeability but more sensitive to the glucose prevention cyanide-induction. In the presence of cyadine, the uptake rate of [³H] colchicine by CH^RC4 cells increased by about 100-fold and approached a rate similar to that of wild-type cells. These results suggest that the colchicine-resistant mutants may be altered in their energy-dependent modulation of drug permeability.     

STUDIES ON POLLEN GERMINATION OF CERTAIN CUCURBITACEAE

Vasil , I. K. (Delhi U., Delhi, India.) Studies on pollen germination of certain Cucurbitaceae. Amer. Jour. Bot. 47(4) : 239—247. Illus. 1960.—Pollen grains of 8 members of the family Cucurbitaceae were cultured in artificial nutrient media. Among the sugars, sucrose proved to be the best for germination of pollen. Although growth regulators, vitamins, antibiotics and some other chemical substances also improved germination of pollen and length of pollen tubes, the effect of boric acid (0.005—0.02%) and borax (0.01%) was most outstanding. The role of sugars and boron in the germination of pollen is discussed.     

Storage of Pollen Grains in Organic Solvents: Effect of Organic Solvents on Leaching of Phospholipids and its Relationship to Pollen Viability

In vitro germinability and membrane integrity (as revealed bythe fluorochromatic reaction (FCR) test) of pollen grains ofCrotalaria retusa L. stored in various organic solvents forsix months at –20±2 °C were studied and correlatedwith leaching of lipids, phospholipids, sugars and free aminoacids from pollen grains into organic solvents during storage.Pollen grains stored in organic solvents with low dielectricconstants (a measure of their non-polar nature), such as hexane,cyclohexane and diethyl ether, showed high scores for germinationand FCR and very little leaching of phospholipids, sugars andamino acids. Pollen grains stored in solvents with high dielectricconstants (a measure of their polar nature) such as isopropanoland methanol did not show germination or positive FCR scores,but showed extensive leaching of phospholipids, sugars and freeamino acids. The viability of pollen grains stored in organicsolvents seems to be determined largely by the effect of theorganic solvents on pollen phospholipid composition, which inturn affects membrane integrity and consequently pollen viability. Crotalaria retusa, organic solvents, pollen storage, viability, phospholipids     

The Metabolism of Oat Leaves during Senescence: I. Respiration, Carbohydrate Metabolism, and the Action of Cytokinins

When the detached first leaves of green or etiolated oat (Avena sativa cv. Victory) seedlings senesce in the dark, their oxygen consumption shows a large increase, beginning after 24 hours and reaching a peak of up to 2.5 times the initial rate by the 3rd day. This effect takes place while the chlorophyll of green leaves, or the carotenoid of etiolated leaves, is steadily decreasing. Kinetin, at a concentration which inhibits the decrease in pigment, completely prevents the respiratory rise; instead, the oxygen consumption drifts downwards. Lower kinetin concentrations have a proportional effect, 50% reduction of respiration being given by about 0.1 mg/l. About one-fifth of the respiratory rise may be attributed to the free amino acids which are liberated during senescence; several amino acids are shown to cause increases of almost 50% in the oxygen consumption when supplied at the concentrations of total amino acid present during senescence. A smaller part of the rise may also be due to soluble sugars liberated during senescence, largely coming from the hydrolysis of a presumptive fructosan. The remainder, and the largest part, of the increase is ascribed to a natural uncoupling of respiration from phosphorylation. This is deduced from the fact that dinitrophenol causes a similar large rise in the oxygen consumption of the fresh leaves or of leaf segments kept green with kinetin, but causes only a very small rise when the oxygen consumption is near its peak in senescent controls. The respiration of these leaves is resistant to cyanide, and 10 mm KCN even increases it by some 30%; in contrast, etiolated leaves of the same age, which undergo a similar rise in oxygen consumption over the same time period, show normal sensitivity to cyanide. The respiratory quotient during senescence goes down as low as 0.7, both with and without kinetin, though it is somewhat increased by supplying sugars or amino acids; glucose or alanine at 0.3 m bring it up to 1.0 and 0.87, respectively.     

Role of Steroids and Amino Acids in Pollen Germination of Eichhornia crassipes Solms.

Detection of amino acids from the pollen grains of Eichhornia crassipes together with isolation of β-sitosterol, stigmasterol and free sugars from the pistil were carried out. The role of the growth potential of the pollen and the growth-influencing compounds of the pistil are discussed. Amino acids and free sugars were detected in pollen and in pistils by paper chromatography. The neutral fractions of the alcohol extract (95%) of the pistil on column chromatography [petroleum ether: benzene (2:1)] gave a crystalline compound (m.p. 136-139°C) with positive Liebermann-Burchard test for sterol. A mass spectrum of the compound showed a mixture of β-sitosterol and stigmasterol, which was confirmed by comparative thin-layer chromatography and gas-layer chromatography.     

Mitochondrial dysfunction mediated by cytoplasmic acidification results in pollen tube growth cessation in Pyrus pyrifolia

The length of pollen tubes grown in synthetic media is normally shorter than those grown in vivo. However, the mechanism(s) underlying the cessation of pollen tube growth under culture conditions remain(s) largely unknown. Here, we report a previously unknown correlation between vacuolar function and the cell's ability to sustain mitochondrial functions in pear pollen tubes. The pear pollen tubes in vitro grew slowly after 15 hours post‐cultured (HPC) and nearly ceased growth at 18 HPC. There was increased malondialdehyde content and membrane ion leakage at 15 HPC compared with 12 HPC. Furthermore, cytoplasmic acidification mainly mediated by decreased vacuolar H⁺‐ATPase [V‐ATPase, Enzyme Commission (EC) 3.6.1.3] activity was observed in pollen tubes after 15 HPC, and this further resulted in mitochondrial dysfunction, including mitochondrial structure disruption, mitochondrial membrane potential collapse and decreases in both oxygen consumption and ATP production. Our findings suggest that vacuoles and mitochondria intimately linked in regulating pollen tube elongation.