A new estimation method for plant cell viability by determining electron transport activity

A new method with which to estimate the viability of tissue-cultured plant cells was developed. In this method, electrons from the electron transport system of Coptis japonica cells were trapped by artificial electron acceptors, and the color of the reduced acceptor was monitored with a spectrophotometer through an optical fiber as surface-reflected light. Cell viability is represented by the amount of increased reflected light per unit time as electron transport activity (ETA). The electron transport activities of cultured Coptis japonica cells that had been effected in viability by the addition of different concentrations of a microbial broth, were related to the ability of the cells to proliferate. When various microbial broths were added to our Coptis japonica cultures, there was a negative correlation between electron transport activity and the amount of berberine released. During usual subculture, electron transport activity increased from the onset of culture, reached a maximum in the late log phase, then decresed rapidly.     

Direct observations on generative cells isolated from pollen grains of Haemanthus katherinae baker

Generative cells from mature pollen grains of Haemanthus katherinae Baker (African blood lily) were isolated by means of a simple squash method and observed by differential interference contrast (DIC), fluorescence and polarizing microscopy. The isolated cells appeared structurally similar to those observed in vivo and gave no evidence of a typical cell wall. Their viability was confirmed using the fluorescein diacetate test. The cell shape changed rapidly as the sucrose concentration of the medium was varied. The squash method of isolating generative cells holds promise for the direct and experimental study of these cells, especially in the living state.Abbreviations FDA Fluorescein diacetate - PAS Periodic-acid-Schiff - DIC differential interference contrast - NA numerical aperture     

Specific differences in tolerance to exogenous berberine among plant cell cultures

The tolerance of plant cells to exogenously administered berberine, an antimicrobial isoquinoline alkaloid, was studied using berberine-producing and nonproducing cell suspension cultures. Both Coptis japonica and Thalictrum flavum cells, which have an intrinsic ability to synthesize berberine, took up exogenous berberine from the culture medium by an energy-requiring active transport to accumulate it exclusively in vacuoles. By contrast, T. minus cells, which excrete indigenous berberine mostly into the medium, did not take up exogenously supplied berberine, indicating that the alkaloid transport in this species is unidirectional. No inhibition of cell growth by exogenous berberine was observed in the three berberine-producing cell cultures. On the other hand, a small amount of exogenous berberine strongly inhibited cell growth in the berberine-free cultures of Datura innoxia, Catharanthus roseus, and Paeonia albiflora. The berberine taken up actively by Datura cells could not be transported into vacuoles but was dispersed in the cytoplasm, causing a severe inhibition of cell growth.     

Biotransformation of cycloalkanones: Controlled oxidative and reductive bioconversions by anAcinetobacter species

Summary Culture conditions have been optimised to enable resting cell cultures ofAcinetobacter calcoaceticus NCIMB 9871 to selectively undertake either oxidative or reductive biotransformations of various bicyclic ketones.     

Ascorbate-phenazine methosulfate-dependent membrane energization in respiratory chain mutants of Escherichia coli

Ascorbate with phenazine methosulfate was able to energize the membrane of inside-out membrane vesicles from cytochrome-containing but not cytochrome-deficient cells of the E., coli, hem A^? mutant SASX76 as measured by the quenching of the fluorescence of acridine dyes. This substrate could also energize vesicle membranes from the ubiquinone-deficient mutant E., coli AN59 in the absence of exogenous ubiquinone. These results suggest that there is site of membrane energization coupled to substrate oxidation in the respiratory chain of E., coli in the cytochrome region between ubiquinone and oxygen.     

Photosynthetic ability in dark-grown Reboulia hemisphaerica and Barbula unguiculata cells in suspension culture

Suspension cultured cells of the liverwort, Reboulia hemisphaerica and of the moss, Barbula unguiculata were independently subcultured in the medium containing 2% glucose in the dark or in the light for more than one year, and the photosynthetic activities of the final cultures were determined. Throughout the culture period light-grown cells of both species contained high amount of chlorophyll (4 to 34 g mg^–1 dry weight) and showed a high photosynthetic activity (10 to 84 mol O₂ mg^–1 chlorophyll h^–1). Dark-grown cells of R. hemisphaerica showed the same level of chlorophyll content and photosynthetic O₂ evolving activity as light-grown cells. Although chlorophyll content in dark-grown B. unguiculata cells was ten-fold lower than that in light-grown cells, the photosynthetic activity of these dark-grown cells was higher than that of light-grown cells based on chlorophyll content.     

Preparation,Characterization and Reactivity of Diastereomeric Sulfoxides of 8,2′-S-Anhydroadenosine

Abstract

The oxidation of 8,2′-S-anhydroadenosine (1a) has been investigated. The major product from the oxidation of 1a using 1-chlorobenzotriazole was the R-sulfoxide. The oxidation of 3′,5′-di-O-acetyl-8,2′-S-anhydroadenosine (1b) gave predominately the S-sulfoxide. These sulfoxides were found to be very succeptible to nucleophilic attack at C-8.     

Multilevel regulation of autophagosome content by ethanol oxidation in HepG2 cells

Acute and chronic ethanol administration increase autophagic vacuole (i.e., autophagosome; AV) content in liver cells. This enhancement depends on ethanol oxidation. Here, we used parental (nonmetabolizing) and recombinant (ethanol-metabolizing) Hep G2 cells to identify the ethanol metabolite that causes AV enhancement by quantifying AVs or their marker protein, microtubule-associated protein 1 light chain 3-II (LC3-II). The ethanol-elicited rise in LC3-II was dependent on ethanol dose, was seen only in cells that expressed alcohol dehydrogenase (ADH) and was augmented in cells that coexpressed cytochrome CYP2E1 (P450 2E1). Furthermore, the rise in LC3-II was inversely related to a decline in proteasome activity. AV flux measurements and colocalization of AVs with lysosomes or their marker protein Lysosomal-Associated Membrane Protein 1 (LAMP1) in ethanol-metabolizing VL-17A cells (ADH⁺/CYP2E1⁺) revealed that ethanol exposure not only enhanced LC3-II synthesis but also decreased its degradation. Ethanol-induced accumulation of LC3-II in these cells was similar to that induced by the microtubule inhibitor, nocodazole. After we treated cells with either 4-methylpyrazole to block ethanol oxidation or GSH-EE to scavenge reactive species, there was no enhancement of LC3-II by ethanol. Furthermore, regardless of their ethanol-metabolizing capacity, direct exposure of cells to acetaldehyde enhanced LC3-II content. We conclude that both ADH-generated acetaldehyde and CYP2E1-generated primary and secondary oxidants caused LC3-II accumulation, which rose not only from enhanced AV biogenesis, but also from decreased LC3 degradation by the proteasome and by lysosomes.     

Accumulation of cyclic guanosine 3':5'-monophosphate in the culture medium of growing cells of Escherichia coli.

In an exponentially growing culture of E. coli, the concentration of cyclic guanosine 3′:5′-monophosphate (cyclic GMP) was found to increase in parallel with the bacterial growth. As the cells approach the stationary phase of growth, the increment of cyclic GMP also ceases progressively to reach to a plateau. When cells are separated from the medium by centrifugation, almost all of the cyclic GMP is recovered in the culture supernatant. The amount of cyclic GMP accumulated is proportional to the number of cells present in the culture. These results suggest that a constant number of cyclic GMP molecules is synthesized each generation of E. coli, and is excreted from the cells to accumulate into the medium.     

Biological oxidation of thianthrene,thioxanthene and dibenzothiophene by the thermophilic organismSulfolobus acidocaldarius

Summary Microbial oxidation of some model aromatic organic sulfur compounds such as thianthrene, thioxanthene and dibenzothiophene by the thermophilic organismSulfolobus acidocaldarius has been studied. Sulfate ions released as an oxidation product were measured to quantify the oxidations. The oxidation of the aforementioned refractory aromatic sulfur compounds byS. acidocaldarius may have applications in organic sulfur removal from hydrocarbon fuels such as coal and oil.     

Fermentation capabilities ofZymomonas mobilis glycolytic enzymes

Summary Cell-free extracts ofZymomonas mobilis were capable of fermenting glucose to ethanol and CO₂ when stimulated by arsenate to act as an ATP uncoupler. 2M glucose was completely converted resulting in a final concentration of 16.5 % w/v ethanol. 1 M glucose was completely converted at temperatures up to 50°C. The results demonstrate that the glycolytic enzymes are more resistant to temperature and ethanol than are the living cells.     

Dynamic dielectrophoretic levitation of living individual cells

The levitation of lone live cells by means of dielectrophoretic force provides a means of determining the relative polarization of the cells and their aqueous support medium. When done over a range of frequencies, a spectrum of dielectric (polarization) responses is obtained which serves to characterize a single living ell. In this manner, individual cells of several microorganisms,Saccharomyces cerevisiae andNetrium digitus, were investigated in the frequency range 10² to 10⁶ Hz. These and related prior studies showed that the positive dielectrophoresis, i.e. where the suspended particle has a greater net polarization than the suspending medium, can be — used to show subtle differences between species, and even between cells of the same culture when using this technique.     

Source of pyrrole-2-carboxylate in mammalian urine

Pyrrole-2-car?ylate, earlier reported in human urine and labeled in rat urine after administration of radioactive proline, arises more directly from labeled hydroxyproline. Antibiotic treatment appeared to exclude epimerization of administered hydroxy-L-proline to a D-epimer by intestinal bacteria. A likely reaction for the in vivo conversion is hydroxy-L-proline oxidation by the L-amino acid oxidase of rat kidney, demonstrable with purified enzyme. Crystalline D-amino acid oxidase also catalyzes a slow oxidation of hydroxy-L-proline. These two reactions are adequate to account for the normal excretion of pyrrole-2-car?ylate by a number of species.     

Kinetics of enzyme release from breadmaking yeast cells in a bead mill

Summary Four intracellular enzymes from two species of breadmaking yeasts- S. cerevisiae and C. boidinii- have been measured as a function of time during its disruption using a bead mill in batch operation. The amount and rate of enzyme released was dependent on its location inside the cell as well as on the kind of yeast. The maximum amount of invertase, a-D-glucosidase, alcohol dehydrogenase and fumarase was obtained at 2,5,10,15 min. respectively for S. cerevisiae. C. boidinii did not show either invertase nor a-D glucosidase activity and the maximum amount of alcohol dehydrogenase and fumarase were reached at 5 and 20 min. respectively.     

Grape pomace: A novel substrate for microbial production of citric acid

Summary Grape pomace was used as substrate for microbial production of citric acid. Of the five cultures examined,Aspergillus niger NRRL 567 was found to produce the greatest amount of citric acid from grape pomace in the presence of methanol at a concentration of 3% (vol/wt). The yield was 60% based on the amount of fermentable sugar consumed.     

Interphase nuclear structure and heterochromatin in Phaseolus plant species

Interphase nuclear structure was studied in five Phaseolus plant species. All the species showed chromocentric nuclear organization in both meristematic and differentiated cells. The number of chromocenters appeared to be a species-specific character. Percentage heterochromatin values, as determined by using three different staining techniques, were higher in meristematic cells than those in differentiated cells. There was a close correspondence between percentage heterochromatin and amount of highly repetitive DNA implying a possible involvement of the latter in chromatin condensation.NCL Communication No. 3381To whom all the correspondence should be addressed.     

Evidence for glycosylation on a DNA-binding protein of <Emphasis Type="Italic">Salmonella enterica</Emphasis>

Background  

All organisms living under aerobic atmosphere have powerful mechanisms that confer their macromolecules protection against oxygen reactive species. Microorganisms have developed biomolecule-protecting systems in response to starvation and/or oxidative stress, such as DNA biocrystallization with Dps (DNA-binding protein from starved cells). Dps is a protein that is produced in large amounts when the bacterial cell faces harm, which results in DNA protection. In this work, we evaluated the glycosylation in the Dps extracted from Salmonella enterica serovar Typhimurium. This Dps was purified from the crude extract as an 18-kDa protein, by means of affinity chromatography on an immobilized jacalin column.     

Comparative studies of the phytochrome system in cell cultures from different plants

Phytochrome contents have been assayed in vivo in cell suspension cultures of Petroselinum hortense, Daucus carota and Glycine max. After transferring the cells to fresh medium phytochrome increased in parallel with the increase in cell number, whereas the amount of phytochrome per cell remained constant. The rate of phytochrome reaccumulation after pretreatment with 15 h red light was very similar in all three systems (2.8–3.6 (e) 10^–5/h). Dark reversion and a fast and slow P_fr destruction were observed in all systems. The rate constants of these reactions varied strongly between the systems. The phytochrome systems of the cell cultures were compared with those of etiolated and light-grown seedlings and it was concluded that the cell suspension cultures of Petroselinum hortense and Daucus carota behaved similarly to light-grown seedlings. In contrast, those of Glycine max behaved similarly to a dark grown seedling.Abbreviations P_r'fr red, far-red absorbing forms of phytochrome - P_tot P_r+P_fr total amount of phytochrome - fwt fresh weight     

Silver toxicity to ferrous iron and pyrite oxidation and its alleviation by yeast extract in cultures ofThiobacillus ferrooxidans

Summary Ferrous-ion oxidation byThiobacillus ferrooxidans was inhibited by 10^–6 M Ag⁺ while a slight inhibition of growth was apparent with 10^–7 M Ag⁺. The threshold toxic concentration was the seme for four different test strains. While prolonged lag phases resulted from culture exposure to Ag⁺, Fe²⁺ oxidation rates after the onset of growth showed little variation under these conditions. Yeast extract (0.02%) partially alleviated the toxicity of silver-ion by reducing the lag periods. Pyrite oxidation byT. ferrooxidans and mixed cultures of acidophiles was tested at 8.3×10^–7 to 8.3×10^–5 M Ag⁺. Strong inhibition was apparent at 8.3×10^–5 M Ag⁺ and little to no inhibition was observed at 8.3×10^–7 M Ag⁺.     

Active cation transport and Na+K+Mg ATPase of the monotreme erythrocytes

The erythrocytes of the echidna (Tachyglossus aculeatus) and platypus (Ornithorhynchus anatinus), which are practically devoid of intracellular ATP content (1), were examined for active Rb⁸⁶ influx and for the presence of Na+K+Mg ATPase. We found that intact erythrocytes of both species possess the ability to actively transport cations. Ouabain sensitive Rb⁸⁶ influx in the echidna was approximately 0.17 μmoles/ml cells × hr, whereas the platypus exhibited a higher value of 0.43 μmoles/ml cells × hr. Surprisingly, ouabain sensitive Na+K+Mg ATPase activity of isolated membranes was high amounting to some 15 to 25 fold higher than the human erythrocyte counterpart determined under identical conditions. These findings suggest that a trace amount of ATP is sufficient to maintain active cation transport across the monotreme cell membranes.