Purification and Properties of Lytic, β-1, 3-Glucanase from Flavobacterium dormitator var. glucanolyticae

An endo β-1, 3-glucanase which is able to disrupt the cells of living yeast has been purified in homogeneous state from the culture filtrate of Flavobacterium dormitator var. glucanolyticae. The molecular weight of the enzyme was estimated to be 17,000 ~ 22,000. The mode of enzyme action has been suggested to be a “random” type of β-1, 3-glucanase. The enzyme preferes larger chains saccharides as substrate for its action, however, smaller oligosaccharides such as laminaritriose and laminaribiose are also decomposed by the enzyme. The Km values of the enzyme for laminarin, laminarihexaose, and laminaritetraose were determined to be 0.26, 1.18, and 2.00 g/liter, respectively. The ability of this enzyme to disrupt the cells of living yeast is its remarkable point, since endo β-1, 3-glucanase of a smaller oligosaccharide-producing type from most sources has been recognized to be inactive (or very weakly active) on living yeast cells.     

Expression and Secretion of Scytalidopepsin B,an Acid Protease from Scytalidium lignicolum,in Yeast

An expression and secretion system for scytalidopepsin B, an acid protease from Scytalidium lignicolum, was constructed in yeast. Saccharomyces cerevisiae AH22 was transformed with an yeast-E. coli shuttle vector, pAM82, in which an yeast invertase signal segment and the cDNA encoding the pro- and mature enzyme regions were inserted. The transformant was found to secret a pepstatin-insensitive acid protease, when cultured aerobically in a low phosphate (Pi) medium. Amino terminal amino acid sequencing analysis indicated that the recombinant acid protease was accurately processed and secreted as a mature form.     

Developmental and environmental influences in the production of a single NAD-dependent fermentative alcohol dehydrogenase by the zygomycete Mucor rouxii

A soluble NAD-dependent alcohol dehydrogenase (ADH) activity was detected in mycelium and yeast cells of wild-type Mucor rouxii. In the mycelium of cells grown in the absence of oxygen, the enzyme activity was high, whereas in yeast cells, ADH activity was high regardless of the presence or absence of oxygen. The enzyme from aerobically or anaerobically grown mycelium or yeast cells exhibited a similar optimum pH for the oxidation of ethanol to acetaldehyde (∼pH 8.5) and for the reduction of acetaldehyde to ethanol (∼pH 7.5). Zymogram analysis conducted with cell-free extracts of the wild-type and an alcohol-dehydrogenase-deficient mutant strain indicated the existence of a single ADH enzyme that was independent of the developmental stage of dimorphism, the growth atmosphere, or the carbon source in the growth medium. Purified ADH from aerobically grown mycelium was found to be a tetramer consisting of subunits of 43 kDa. The enzyme oxidized primary and secondary alcohols, although much higher activity was displayed with primary alcohols. K _m values obtained for acetaldehyde, ethanol, NADH₂, and NAD⁺ indicated that physiologically the enzyme works mainly in the reduction of acetaldehyde to ethanol. Received: 11 March 1999 / Accepted: 14 July 1999     

FTIR and UV spectroscopy in real-time monitoring of S. cerevisiae cell culture

A combination of FTIR and UV spectroscopy is proposed as a novel technique for integrated real-time monitoring of metabolic activity and growth rates of cell cultures, required for systematic studies of cellular low-frequency (LF) electric and magnetic field (EMF) effects. As an example, we investigated simultaneous influence of periodic LF 3D EMFs on a culture of Saccharomyces cerevisiae (baker's yeast) cells. Amplitudes, frequencies and phases of the field components were the variable parameters. Electromagnetic fields were found to efficiently control the activity of the yeast cells, with the resulting CO₂ production rates, as monitored by FTIR spectroscopy, varying by at least one order of magnitude due to the field action. Additionally, population dynamics of the yeast cells was monitored by UV absorption of the yeast culture at λ_prob = 320 nm, and compared to the CO₂ production rates. The detected physiologically active frequencies are all below 1 kHz, namely, 800 Hz excitation was effective in reducing the metabolic rates and arresting cell proliferation, whereas 200 Hz excitation was active in accelerating both cell proliferation and overall metabolic rates. The proposed methods produce objective, reliable and quantitative real-time results within minutes and may be used in various tasks that could benefit from a rapid feedback they provide in the form of metabolic and growth rates. Amplitude and frequency dependences of the LF EMF effects from individual field components with different polarizations were recorded and qualitatively interpreted based on a simple model, describing ion diffusion through a membrane channel.     

Tetrazolium reduction in Saccharomyces cerevisiae

Summary The tetrazolium salt, 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) was used to determine viable respiring cells in batch cultures of Saccharomyces cerevisiae. Respiring cells reduce INT to water insoluble iodonitrotetrazolium formazan (INT-formazan) which is deposited within the respiring cell. The INT-formazan granules can be observed by brightfield microscopy. This allows a rapid quantitative determination of the percentage of respiring cells and total cells within the same microscopic field.In actively growing batch cultures of S. cerevisiae, the respiring cell count was equal to the total cell count for the first 72 h of the growth cycle. After 144 h of incubation only 22.7% of the total cell numbers were actively respiring.     

A comparison of methods for assessing yeast viability

Summary Eight different methods were used to assess the cell viability of four strains of Saccharomyces. Staining with Mg-ANS, primuline yellow, FITC and methylene blue gave a good index of yeast cell viability. The standard plate count technique and microcolony formation also gave a good measure of cell viability. Fluorescent staining with acridine orange was the least useful of the methods tested. INT dye reduction gave a good index of respiring cells depending upon the yeast strain tested.     

Isolation of giant mitochondrial nucleoids from the yeastSaccharomyces cerevisiae

Summary The yeast cellsSaccharomyces cerevisiae grown up to stationary phase under either anaerobic conditions, or aerobic conditions in the presence of a respiratory inhibitor, antimycin A, had distinctive giant mitochondrial nucleoids (mt-nucleoids) (apparent diameter 0.6–0.9 m) in contrast with the small mt-nucleoids (apparent diameter 0.2–0.4 m) in respiratory-sufficient cells grown aerobically, as revealed by DAPI-fluorescence microscopy. The cytoplasmic respiratory-deficient cells (rho^– cells), which were induced by treatment of wild-type cells with ethidium bromide, showed both giant and small mt-nucleoids of irregular size. In order to examine the structural and functional differences between giant and small mt-nucleoids, the former were successfully isolated from spheroplasts of three different cells by differential centrifugation and centrifugation on a discontinuous sucrose gradient. The isolated giant mt-nucleoids were intact in the morphology and were free of significant contamination by nuclear chromatin. The number of protein components involved in each of three different giant mt-nucleoids was similar to the number in small mt-nucleoids from aerobically grown cells, though a few noticeable differences were also recognized. DNA-binding proteins with molecular masses of 67 kDa, 52 kDa, 50 kDa, 38 kDa, 26 kDa, and 20 kDa were the main components of small mt-nucleoids from aerobically grown cells as detected by chromatography on native DNA-cellulose. In contrast, the 67 kDa and 52 kDa proteins were hardly detected in corresponding fractions of giant mt-nucleoids from anaerobically grown cells and from rho^– cells grown aerobically. On the other hand, mt-nucleoids from aerobically grown cells in the presence of antimycin A seemed to lack the 67 kDa protein but to have a small amount of the 52 kDa protein. This is the first demonstration of the variance of protein species involved in yeast mt-nucleoids according to the respiratory activity of mitochondria.     

Qualitative Behavior of Spontaneous Potentials from Explants of 15 Day Chick Embryo Telencephalon in Vitro

The apparatus and technique used in the preparation and observation of explants of brain tissue capable of producing spontaneous potentials in vitro are described. The magnitude and pattern of spontaneous potentials from explants of telencephalon of 15 day chick embryos (measured using external bare platinum electrodes) and some aspects of their "normal" behavior during 12 days in vitro are also described. No change was noted in these potentials with change of amplifiers, recorders, or electrodes. The response of the potentials to change in temperature and proportionate composition of the atmosphere around the explant was such as to suggest that the potentials arise as a result of a living process. The changes brought about by the administration of anesthetics, strychnine, brucine, and barbiturates were those that might be anticipated in a normal functional activity of the central nervous system. It is concluded that these potentials are a true physiological phenomenon and arise from living cells of the central nervous system.     

Accumulation of magnesium ions during fermentative metabolism in Saccharomyces cerevisiae

When cells of Saccharomyces cerevisiae were grown aerobically under glucose-repressed conditions, ethanol production displayed a hyperbolic relationship over a limited range of magnesium concentrations up to around 0.5 mM. A similar relationship existed between available Mg²⁺ and ethanol yield, but over a narrower range of Mg²⁺ concentrations. Cellular demand for Mg²⁺ during fermentation was reflected in the accumulation patterns of Mg²⁺ by yeast cells from the growth medium. Entry of cells into the stationary growth phase and the time of maximum ethanol and minimum sugar concentration correlated with a period of maximum Mg²⁺ transport by yeast cells. The timing of Mg²⁺ transport fluxes by S. cerevisiae is potentially useful when conditioning yeast seed inocula prior to alcohol fermentations. Received 04 March 1996/ Accepted in revised form 21 August 1996     

Light-flash analysis of the photoenzymic repair process in yeast cells III. The photoenzymic repair of respiratory-sufficient and respiratory-deficient cells cultured aerobically and anaerobically

In the previous papers, determinations of the number of deoxyribodipyrimidine photolyase or photoreactivating enzyme (PRE) molecules per cell and of the rate constant for the formation of the PRE-pyrimidine dimer complex, k₁, for yeast cells cultured aerobically, were described. In the present work, the number of PRE molecules and ther ate constant k₁ for respiratory-sufficient (RS) and respiratory-deficient (RD) cells of yeast cultured anaerobically were determined. The numbers of PRE molecules in both RS and RD cells cultured anaerobically were larger than those in cells cultured aerobicaly, whereas the rate constant k₁ (nuclear volume). (PRE molecules)^?1 sec^?1 showed a reverse relationship. The numbers of PRE molecules in cells cultured aerobically in various media were also determined.     

Temperature induced atypical morphogenesis of the obligately psychrophilic yeast,Leucosporidium stokesii

The obligately psychrophilic yeast, Leucosporidium stokesii increased in size, produced irregular wall growth and formed atypical buds when incubated within one to three degrees above 20 °C, the maximum growth temperature. Incubation of cells anaerobically or aerobically in the presence of 2,4-dinitrophenol at the elevated temperatures prevented the development of atypical buds. An investigation of subcellular morphology revealed that the atypical bud was anucleate, did not form a septum between bud and parent cell and produced numerous cytoplasmic vesicles. On shift-down to 15 °C, the optimum growth temperature, nuclear division, migration and septum formation resumed.     

A sensitive method for measuring oxygen consumption

A technique is presented whereby oxygen consumption rates of the order of 10⁻⁶ μl/hr can be measured, thus providing a means for studying the respiration rates of single cells, even slowly respiring ones. The technique is based on the principle of incubating cells in extremely small chambers containing highly concentrated hemoglobin solutions. The absorbance shift occurring when hemoglobin is transformed from its oxygenated to its deoxygenated form is recorded microspectrophotometrically. The results obtained by this technique seem to be well in accordance with those of the Warburg manometric method. The technique is convenient and easy to handle and the sensitivity can be varied within wide limits, permitting different types of materials to be studied. In experiments using yeast cells, respiration rates from 1.0 × 10⁻⁶ to 1.8 × 10⁻⁶ μl O₂/cell/hr were revealed.     

Electric and hydrodynamic properties of polypeptides in solution. II. Conformation of poly(L-glutamic acid) in various organic solvents

The electric birefringence of poly(L -glutamic acid) (PLGA) in methanol, dimethyl sulfoxide, dimethylformamide, N-methylacetamide, trifluoroacetic acid, dioxane–water mixtures (3:1 and 4:1 by volume), and dioxane–formamide mixture (1:1 by volume) has been measured by the use of the rectangular pulse technique at 30 °C. The intrinsic viscosity has also been measured at the same temperature. The magnitude of the specific Kerr constant and the intrinsic viscosity suggests that PLGA is helical and has a large dipole moment in methanol, dimethyl sulfoxide, dimelhylformamide, N-methylacetamide, and dioxane–water mixtures. In this case we have obtained the length distribution curve and the mean length of PLGA molecules from the decay of the electric birefringence, by applying the method recently developed for helical polypeptides. Furthermore, we have proposed and applied a method of obtaining the mean dipole moment and the optical anisotropy factor from the field strength dependence of the electric birefringence for polydisperse systems on the basis of the knowledge on the length distribution. The results show that PLGA may have a different helical conformation in dimethyl sulfoxide. The specific Kerr constant of PLGA in trifluoroacetic acid is very small, which suggests that PLGA is a random coil in this solvent.     

Disappearance of Phosphorylation Activity of Nucleotides from the Mitochondria-rich Cells of an Yeast,Hansenula jadinii; a Modified Pasteur Effect

An yeast, Hansenula jadinii, which was one of the best producers of CDP-choline on our system, lost its activity when cultured in jar fermenter. This phenomenon was also reproduced in flasks. Cells cultured aerobically in the medium containing 1 % of glucose (A-cells) could not phosphorylate nucleotides although development of mitochondria was observed, whereas cells cultured less aerobically in the medium containing 5% of glucose (D-cells) could phosphorylate CMP to CTP and finally produce CDP-choline although they had only poor mitochondria. Further study revealed that the A-cells were unstable in hexokinase activity, although they had the dense cytosol, whereas the D-cells remained stable, and they had many round particles. Glycolytic activity was about 4 times stronger in the D-cells than in the A-cells. The phenomenon that respiration (development of mitochondria) suppressed fermentation (glycolysis) has been known as the Pasteur effect. However, in our system, phosphofructokinase, the primary key enzyme of the Pasteur effect, was active in the A-cells. Therefore, our phenomenon seemed to be a modified Pasteur effect.     

Selective activity of <Emphasis Type="Italic">Mucor plumbeus</Emphasis> reductase towards (−)-camphorquinone

The biotransformation of 1R-(−)-camphorquinone, achieved by growing cells of four fungi species isolated from soil (Mucor plumbeus, Lecanicillium muscarium, Thamnostylum sp. and Syncephalastrum racemosum), was investigated in optimized culture media for each species. Fungi were grown aerobically under shaking and their activities with respect to camphorquinone were monitored for 20 days by gas chromatography coupled to mass spectrometry (GCMS). Camphorquinone was found to be stable in control flasks throughout the experiment. The most interesting results were found for M. plumbeus, which was only able to perform monoreduction of camphorquinone when cultivated on a glucose–peptone–yeast extract medium. Large-scale experiments were set up and the camphorquinone biotransformation products formed by M. plumbeus were purified by column chromatography and identified by ¹H and ¹³C nuclear magnetic resonance (NMR). Theoretical calculations were employed as a complementary technique to unambiguously identify the biotransformation products. These findings suggest that M. plumbeus could be of great use for the selective reduction of camphorquinone and related compounds.     

Mapping HA‐tagged protein at the surface of living cells by atomic force microscopy

Single‐molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2‐adrenergic receptor (β2‐AR), a G protein‐coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2‐AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. Copyright © 2014 John Wiley & Sons, Ltd.     

Induction of ELF transmembrane potentials in relation to power-frequency electric field bioeffects in a plant root model system

Summary Seminal roots ofCucumis sativus andCucurbita maxima were exposed to 60 Hz electric fields of 100–500 Vm^–1 in a conducting aqueous inorganic growth medium. Root growth rates were measured to produce a dose-response relationship for each species. The species were selected for study because of their familial relationship, reported sensitivity to 60 Hz, 360 Vm^–1 electric fields, and differing average root cell sizes. The latter characteristic influences the magnitude of ELF membrane potentials induced by constant-strength applied electric fields, but does not affect the magnitude of the electric field strength tangent to the cell surface. The difference in average root cell size betweenC. sativus (smaller cells) andC. maxima (larger cells) was used to evaluate two alternate hypotheses that the observed effect on root growth is stimulated by [1] the electric field tangent to the cell surface, or (2) a field-induced perturbation in the normal transmembrane potential of the cells.The results of the dose-response relationship studies are qualitatively consistent with the hypothesis that the effect is elicited by induced transmembrane potentials. The smaller-celled roots showed a substantially higher response threshold [C. sativus; E ₀ ^TH 330 Vm^–1] than did the larger-celled species [C. maxima; E ₀ ^TH 200 Vm^–1]. At field strengths above the response thresholds in both species, the growth rate ofC. sativus roots was less affected than that ofC. maxima roots exposed to the same field strength.     

Three-dimensional FRET reconstruction microscopy for analysis of dynamic molecular interactions in live cells

Analysis of cellular pathways requires concentration measurements of dynamically interacting molecules within the three-dimensional (3D) space of single living cells. Förster resonance energy transfer (FRET) microscopy from widefield, from confocal, and potentially from superresolution microscopes can access this information; however, these measurements are distorted by the inherent 3D blurring of optical imaging, spectral overlap of fluorophores, and detection noise. We propose a mathematical model of these processes and demonstrate, through simulation, how these distortions limit the dynamic range and sensitivity of conventional FRET microscopy. Using this model, we devise and validate a new approach (called 3D-FRET stoichiometry reconstruction, 3DFSR) for reconstructing 3D distributions of bound and free fluorescent molecules. Previous attempts to reconstruct 3D-FRET data relied on sequential spectral unmixing and deconvolution, a process that corrupts the detection statistics. We demonstrate that 3DFSR is superior to these approaches since it simultaneously models spectral mixing, optical blurring, and detection noise. To achieve the full potential of this technique, we developed an instrument capable of acquiring 3D-FRET data rapidly and sensitively from single living cells. Compared with conventional FRET microscopy, our 3D-FRET reconstruction technique and new instrumentation provides orders of magnitude gains in both sensitivity and accuracy wherein sustained high-resolution four-dimensional (x,y,z,t) imaging of molecular interactions inside living cells was achieved. These results verify previous observations that Cdc42 signaling is localized to the advancing margins of forming phagosomes in macrophages.     

Attractiveness of CO2 released by root respiration fades on the background of root exudates

Plants are endangered at their roots by soil-dwelling rhizophagous insects. These below-ground living herbivores may orient to the source of carbon dioxide (CO₂), an ubiquitous volatile released by respiring plant roots. Here, we studied the interaction of CO₂ and other plant root-derived chemical stimuli with regard to the chemical orientation of the polyphagous larvae of Melolontha melolontha L. (Scarabaeidae). A soil arena was developed that enabled both determination of the actual soil CO₂ concentration and the behavioural response of an insect to (a) CO₂ gradients per se, (b) chemical stimuli released from respiring, undamaged roots of plants potted into vermiculite in this arena and (c) combinations of CO₂ gradients and root-derived stimuli. In a root-free arena, larvae of M. melolontha oriented to the source of synthetic CO₂. However, similar CO₂ gradients generated by host plant roots did not attract the larvae. Neither did a synthetic CO₂ gradient combined with aqueous extracts from rhizospheres with undamaged plant roots elicit an attractive effect. Our data suggest that orientation of cockchafer larvae within CO₂ gradients generated by respiring roots is ‘masked’ by an aqueous extract from a rhizosphere with undamaged roots. The results emphasise that effects of behaviour modifying plant-derived compounds need to be investigated against the background of naturally co-occurring chemicals. The significance of our results for orientation of soil living insects is discussed with respect to abiotic conditions in natural soil and the role of soil microorganisms for the attractiveness of plant roots.     

Thermal noise limit on the sensitivity of cellular membranes to power frequency electric and magnetic fields

Several authors have concluded that thermal electromagnetic noise will be of sufficient magnitude to overwhelm electric and/or magnetic fields induced by environmentally generated, power frequency electric and magnetic fields in the membranes of living cells located in the bodies of humans. Yet, there are research reports that indicate that living cells may respond to power frequency electric and/or magnetic field levels well below the limits set by these thermal noise arguments. The purpose of this study is to suggest that published thermal arguments may not make a full accounting of all membrane force fields of thermal origin, and that when such an accounting is made, the net thermal noise fields may be smaller in the power frequency range than previously thought. If this analysis is correct, there may be no thermal noise barrier that precludes the possibility of cellular membranes of human cells responding to environmental levels of power frequency electric or magnetic fields.