Numerical simulations of the development of an open discharge

Plasma Physics Reports - Results are presented from numerical simulations of the time evolution of open discharges in helium that are excited in the presence of an anode grid and generate electron...     

Spectroscopic measurements of the parameters of the helium plasma jets generated in the plasma focus discharge at the PF-3 facility

The spectroscopic technique used to measure the parameters of the plasma jets generated in the plasma focus discharge and those of the plasma of the immobile gas through which these jets propagate is described. The time evolution of the intensities and shapes of spectral lines in experiments carried out with helium at the PF-3 facility was studied by means of electron-optical streak cameras. The plasma electron temperature, T ≈ 4–5 eV, was determined from the intensity ratio of two spectral lines, one of which (λ₁ = 5876 Å) belongs to neutral helium, while the other (λ₂ = 4686 Å), to hydrogen-like helium ions. The plasma density at different time instants was determined from the Stark broadening of these lines in the electric fields of different nature. The plasma density is found to vary from 4 × 10¹⁴ to 2 × 10¹⁷ cm^?3.     

Decreasing motion artifacts in calcium-dependent fluorescence transients from the perfused mouse heart using frequency filtering

A strategy has been developed for the removal of motion artifact and noise in calcium-dependent fluorescence transients from the perfused mouse heart using frequency filtering. An analytical model indicates that the spectral removal of motion artifacts is independent of the phase shift of the motion waveform in the frequency domain, and thus to the time shift (or delay) of motion in the time domain. This is based on the "shift theorem" of Fourier analysis, which avoids erroneous correction of motion artifact when using the motion signal obtained using reflectance from the heart. Several major steps are adopted to implement this model for elimination of motion as well as detection noise from the fluorescence transient signals from the calcium-sensitive probe Rhod-2. These include (1) extracting the fluorescence calcium transient signal from the raw data by using power spectrum density (PSD) in the frequency domain by subtracting the motion recorded using the reflectance of excitation light, (2) digitally filtering out the random noise using multiple bandpass filters centralized at harmonic frequencies of the transients, and (3) extracting high frequency noise with a Gaussian Kernel filter method. The processed signal of transients acquired with excessive motion artifact is comparable to transients acquired with minimal motion obtained by immobilizing the heart against the detection window, demonstrating the usefulness of this technique.     

Comparison of kinetics of formation of helices and hydrophobic core during the folding of staphylococcal nuclease from acid.

Our previous kinetic study of the acid and base-induced folding/unfolding transitions of staphylococcal nuclease (SNase) has monitored Trp-140 fluorescence. Trp-140 is located near the flexible COOH terminus and whether or not its fluorescence reflects the overall conformation of the protein has yet to be established. Here we show that the fluorescence intensity of Try-140 correlated closely with the thermal stability (i.e., the calorimetric enthalpy, delta Hcal, of unfolding) of the protein in the pH range 7 to 2.5, confirming that it is a good measure of the overall protein structure. Circular dichroism (CD) at 222 nm, which reflects the helical content of the protein molecule, was used to follow the same folding/unfolding transition in order to compare kinetics of the helix formation and of the appearance of the hydrophobic core. In addition to the three kinetic phases reported earlier with the fluorescence detection, there were a rapid reaction (completed within the 25 ms mixing time of the instrument), which comprised 15% of the signal, and a very slow reaction (time constant > 300 s), which comprised 19% of the signal. With the fluorescence detection for the folding from acid, only 5% of the signal occurred in the rapid phase and there was no reaction slower than 300 s. By comparing kinetics of folding at pH 7 by the CD and fluorescence detection methods, we concluded that: (a) Roughly 15% of the helix content of SNase accumulated before significant changes in the hydrophobic environment (< 5%) of Trp-140 could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the oxygen evolution step in plants determined from flash-induced chlorophyll t a fluorescence

Photosystem II (PS II) of plants and cyanobacteria, which catalyzes the light-induced splitting of water and the release of oxygen, is the primary source of oxygen in the earth atmosphere. When activated by short light flashes, oxygen release in PS II occurs periodically with maxima after the third and the seventh flashes. Many other processes, including chlorophyll (Chl) t a fluorescence, are also modulated with period of four, reflecting their sensitivity to the activity of Photosystem II. A new approach has been developed for the analysis of the flash-induced fluorescence of Chl t a in plants, which is based on the use of the generalized Stern–Volmer equation for multiple quenchers. When applied to spinach thylakoids, this analysis reveals the presence of a new quencher of fluorescence whose amplitude is characterized by a periodicity of four with maxima after the third and the seventh flashes, in phase with oxygen release. The quencher appears with a delay of 0.5 ms followed by a rise time of 1.2–2 ms at pH 7, also in agreement with the expected time for oxygen evolution. It is concluded that the quencher is a product of the reaction leading to the oxygen evolution in PS II. The same quenching activity, maximal after the third flash, could be seen in dark adapted leaves, and provides the first fully time-resolved measurement of the kinetics of the oxygen evolution step in the leaf. Thus, the non-invasive probe of Chl t a fluorescence provides a new and sensitive method for measuring the kinetics of oxygen evolution with potential for use in plants and cyanobacteria t in vivo.     

Functional characterization of monomeric photosystem II core preparations from Thermosynechococcus elongatus with or without the Psb27 protein

Two monomeric fractions of photosystem II (PS II) core pacticles from the thermophilic cyanobacterium Thermosynechococcus elongatus have been investigated using flash-induced variable fluorescence kinetics and EPR spectroscopy. One fraction was highly active in oxygen evolution and contained the extrinsic protein subunits PsbO, PsbU, and PsbV. The other monomeric fraction lacked oxygen evolving activity as well as the three extrinsic subunits, but the luminally located, extrinsic Psb27 lipoprotein was present. In the monomeric fraction with bound Psb27, flash-induced variable fluorescence showed an absence of oxidizable Mn on the donor side of PS II and impaired forward electron transfer from the primary quinone acceptor, QA. These results were confirmed with EPR spectroscopy by the absence of the "split S1" interaction signal from YZ* and the CaMn4 cluster and by the absence of the S2-state multiline signal. A different protein composition on the donor side of PS II monomers with Psb27 was also supported by the lack of an EPR signal from cytochrome c550 (in the PsbV subunit). In addition, we did not observe any oxidation of cytochrome b559 at low temperature in this fraction. The presence of Psb27 and the absence of the CaMn4 cluster did not affect the protein matrix around YD or the acceptor side quinones as can be judged from the appearance of the corresponding EPR signals. The diminished electron transport capabilities on both the donor and the acceptor side of PS II when Psb27 is present give further indications that this PS II complex is involved in the earlier steps of the PS II repair cycle.     

Microdissection and painting of the Y chromosome in spinach (Spinacia oleracea)

Spinach has long been used as a model for genetic and physiological studies of sex determination and expression. Although trisomic analysis from a cross between diploid and triploid plants identified the XY chromosome as the largest chromosome, no direct evidence has been provided to support this at the molecular level. In this study, the largest chromosomes of spinach from mitotic metaphase spreads were microdissected using glass needles. Degenerate oligonucleotide primed polymerase chain reaction was used to amplify the dissected chromosomes. The amplified products from the Y chromosome were identified using the male-specific marker T11A. For the first time, the largest spinach chromosome was confirmed to be a sex chromosome at the molecular level. PCR products from the isolated chromosomes were used in an in situ probe mixture for painting the Y chromosome. The fluorescence signals were mainly distributed on all chromosomes and four pair of weaker punctate fluorescence signal sites were observed on the terminal region of two pair of autosomes. These findings provide a foundation for the study of sex chromosome evolution in spinach.     

Fast photoacoustic transients from dark-adapted intact leaves: oxygen evolution and uptake pulses during photosynthetic induction — a phenomenology record

Using a photoacoustic technique it has been possible to observe fast oxygen evolution and uptake transients at a high time resolution (approx. 0.2 s), when a dark-adapted leaf is reilluminated. There is initially a rapid pulse of oxygen evolution, correlated with the initial fluorescence rise (total duration under the experimental conditions used about 1–2 s), corresponding presumably to the photoreduction of the plastoquinone pool. This phenomenon may be utilized to calibrate the oxygen-evolution photoacoustic signal. The first pulse is followed by a series of slower bursts of oxygen uptake and evolution, reflecting various pools which are expressed following sequential activation of various parts of the photosynthetic apparatus, until achievement of a steady state.Abbreviations and symbols RuBP ribulose-1,5-bisphosphate - PSI, PSII photosystems I, II - F_o, F_m, F(t) initial, maximum and instantaneous chlorophyll fluorescence emission     

Release of calcium from intracellular stores in rat basophilic leukemia cells monitored with the fluorescent probe chlortetracycline.

Release of calcium from intracellular stores of rat basophilic leukemia cells was monitored using the fluorescent probe chlortetracycline. The ability of chlortetracycline to indicate release from intracellular calcium stores was initially validated. The decrease of chlortetracycline fluorescence upon antigen-stimulation was not the result of secretion of granule-associated dye or of changes in the properties of the membranes. The chlortetracycline fluorescence signal was not influenced by Ca2+ influx across the plasma membrane. Results obtained from these chlortetracycline fluorescence measurements corresponded well with 45Ca efflux data, an indirect measurement of release of calcium from stores. Chlortetracycline was used to examine the rate of antigen-induced release of calcium from stores, the depletion of intracellular calcium stores by EGTA, and the relationship between the antigen-stimulated release of stored calcium and exocytosis. Chlortetracycline was shown to be a useful qualitative indicator for the release of intracellular calcium with a relatively rapid response time.     

Insights into the function of PsbR protein in Arabidopsis thaliana

The functional state of the Photosystem (PS) II complex in Arabidopsis psbR T-DNA insertion mutant was studied. The DeltaPsbR thylakoids showed about 34% less oxygen evolution than WT, which correlates with the amounts of PSII estimated from Y(D)(ox) radical EPR signal. The increased time constant of the slow phase of flash fluorescence (FF)-relaxation and upshift in the peak position of the main TL-bands, both in the presence and in the absence of DCMU, confirmed that the S(2)Q(A)(-) and S(2)Q(B)(-) charge recombinations were stabilized in DeltaPsbR thylakoids. Furthermore, the higher amount of dark oxidized Cyt-b559 and the increased proportion of fluorescence, which did not decay during the 100s time span of the measurement thus indicating higher amount of Y(D)(+)Q(A)(-) recombination, pointed to the donor side modifications in DeltaPsbR. EPR measurements revealed that S(1)-to-S(2)-transition and S(2)-state multiline signal were not affected by mutation. The fast phase of the FF-relaxation in the absence of DCMU was significantly slowed down with concomitant decrease in the relative amplitude of this phase, indicating a modification in Q(A) to Q(B) electron transfer in DeltaPsbR thylakoids. It is concluded that the lack of the PsbR protein modifies both the donor and the acceptor side of the PSII complex.     

Fluorescence changes from single striated muscle fibres injected with labelled troponin C (TnCDANZ)

A fluorescently labelled derivative of the calcium binding subunit of troponin, TnC, has been injected into isolated striated muscle fibres from the barnacle Balanus nubilus. The Ca2+ affinity of isolated TnC is close to that of intact troponin when located in the thin filament. Excitation of the TnCDANZ within the muscle cell (325nm) revealed a marked fluorescence at 510 nm and was similar to that observed in vitro, which was absent at 400 or 600 nm after subtraction of the fibre autofluorescence. High Ca2+ salines increased the fluorescence at 510 nm by roughly 2 times. Single voltage clamp pulses produced a rapid rise in fluorescence at 510 nm after allowing for any non-specific changes at 400 nm, and this signal preceded force development by approx. 55 ms at 22 degrees C. It reached a maximum at the same time as force and subsequently decayed more slowly. The fluorescence signal increased in magnitude with increase in stimulus intensity. These results suggest that Ca2+ attaches rapidly to the contractile filament, but is lost relatively slowly and imply a slow decay of the activation process.     

Observation of Continuous Contraction and a Metastable Misfolded State during the Collapse and Folding of a Small Protein

To obtain proper insight into how structure develops during a protein folding reaction, it is necessary to understand the nature and mechanism of the polypeptide chain collapse reaction, which marks the initiation of folding. Here, the time-resolved fluorescence resonance energy transfer technique, in which the decay of the fluorescence light intensity with time is used to determine the time evolution of the distribution of intra-molecular distances, has been utilized to study the folding of the small protein, monellin. It is seen that when folding begins, about one-third of the protein molecules collapse into a molten globule state (I_MG), from which they relax by continuous further contraction to transit to the native state. The larger fraction gets trapped into a metastable misfolded state. Exit from this metastable state occurs via collapse to the lower free energy I_MG state. This exit is slow, on a time scale of seconds, because of activation energy barriers. The trapped misfolded molecules as well as the I_MG molecules contract continuously and slowly as structure develops. A phenomenological model of Markovian evolution of the polymer chain undergoing folding, incorporating these features, has been developed, which fits well the experimentally observed time evolution of distance distributions. The observation that the “wrong turn” to the misfolded state has not been eliminated by evolution belies the common belief that the folding of functional protein sequences is very different from that of a random heteropolymer, and the former have been selected by evolution to fold quickly.     

Whole-cell imaging at nanometer resolutions using fast and slow focused helium ions

Observations of the interior structure of cells and subcellular organelles are important steps in unraveling organelle functions. Microscopy using helium ions can play a major role in both surface and subcellular imaging because it can provide subnanometer resolutions at the cell surface for slow helium ions, and fast helium ions can penetrate cells without a significant loss of resolution. Slow (e.g., 10–50 keV) helium ion beams can now be focused to subnanometer dimensions (∼0.25 nm), and keV helium ion microscopy can be used to image the surfaces of cells at high resolutions. Because of the ease of neutralizing the sample charge using a flood electron beam, surface charging effects are minimal and therefore cell surfaces can be imaged without the need for a conducting metallic coating. Fast (MeV) helium ions maintain a straight path as they pass through a cell. Along the ion trajectory, the helium ion undergoes multiple electron collisions, and for each collision a small amount of energy is lost to the scattered electron. By measuring the total energy loss of each MeV helium ion as it passes through the cell, we can construct an energy-loss image that is representative of the mass distribution of the cell. This work paves the way to use ions for whole-cell investigations at nanometer resolutions through structural, elemental (via nuclear elastic backscattering), and fluorescence (via ion induced fluorescence) imaging.     

Simultaneous measurement of oxygen evolution and chlorophyll fluorescence from leaf pieces

An apparatus is described which permits the simultaneous measurement of O₂ evolution and chlorophyll a fluorescence from illuminated discs or pieces of green leaves. O₂ is measured in the gas phase in a temperature-controlled chamber of approximately 5-milliliter capacity. Calibration is effected by injection of air through vents. Response time is approximately 1.5 seconds for O₂, and full scale deflection, in normal operating mode, is approximately 10 micromoles O₂. The apparatus may also be used to monitor fluorescence alone, in an open mode, in which gas is passed continuously through the chamber.     

Characterization by EPR spectroscopy of cytochrome b-562 isolated from the cytochrome b-c1 complex of Rhodopseudomonas sphaeroides R-26

The EPR spectra of cytochrome b-562 isolated from the cytochrome b-c1 complex of Rhodopseudomonas sphaeroides were measured at liquid helium temperature. The purified cytochrome b-562 gives a high spin signal at g = 6.0. Anaerobic titration of this signal confirmed the presence of two redox components with Em = 40 and -110 mV at pH 7.5. These values are consistent with the published ones, Em = 55 and -100 mV at pH 7.0, that were optically estimated for the same type of preparation (Iba et al. (1985) FEBS Lett. 183, 151-154). The power saturation behavior of the g = 6.0 signal at different redox potentials indicated a direct spin-spin interaction between these two redox centers.     

A new label technology for the detection of specific polymerase chain reaction products in a closed tube

A novel signal generation principle suitable for real time and end-point detection of specific PCR products in a closed tube is described. Linear DNA probes were labeled at their 5′-ends with a stable, fluorescent terbium chelate. The fluorescence intensity of this chelate is lower when it is coupled to single-stranded DNA than when the chelate is free in solution. The synthesized probes were used in the real time monitoring of PCR using a prototype instrument that consisted of a fluorometer coupled to a thermal cycler. When the probe anneals to a complementary target amplicon, the 5′→3′ exonucleolytic activity of DNA polymerase detaches the label from the probe. This results in an enhanced terbium fluorescence signal. Since terbium has a long excited state lifetime, its fluorescence can be measured in a time-resolved manner, which results in a low background fluorescence and a 1000-fold signal amplification. The detection method is quantitative over an extremely wide linear range (at least 10–10⁷ initial template molecules). The label strategy can easily be combined with existing label technologies, such as TaqMan 5′-exonuclease assays, in order to carry out multiplex assays that do not suffer from overlapping emission peaks of the fluorophores.     

Steady-state fluorescence emission from the fluorescent probe, 5-iodoacetamidofluorescein, bound to hemoglobin

In the past, fluorescence emission from an extrinsic fluorophore bound to heme-proteins would only be studied with the removal of the heme since fluorescence from the fluorophore could not be detected using right-angle optics. Using front-face fluorometry, a significant steady state emission signal originating from the probe bound to hemoglobin is detected. This is the first report of the detection of extrinsic fluorescence of a probe bound to a heme-protein. We also demonstrate that the extrinsic probe, 5-iodoacetamidofluorescein, is covalently bound to hemoglobin, specifically at beta 93 Cysteine. Ligand binding results in a change in the fluorophore fluorescence intensity as predicted by hemoglobin crystallographic studies. Efficiency of energy transfer measurements are made.     

Fluorescence properties of chloroplasts from manganese deficient and mutant tobacco

1. The kinetics of the fluorescence induction are described for chloroplasts from normal green tobacco, from the aurea tobacco mutant Su/su, from the photosynthetically inactive yellow patches of a variegated tobacco, and from tobacco plants grown in absence of manganese. The first two types display the well-known biphasic induction, but the Su/su chloroplasts have a distinctly slower rise time. Manganese deficient chloroplasts show a significantly higher fluorescence yield than any other type of chloroplasts studied. The kinetics of their fluorescence, on the other hand, are similar to those observed with the inactive chloroplasts from the variegated tobacco: the fluorescence rise is small, and the fluorescence yield is not changed very much by the addition of a reducing agent like hydrosulfite, or by addition of an oxidant like ferricyanide, or by an inhibition of the electron flow in Photosystem II with 3(3,4-dichlorophenyl)-1,1-dimethylurea.

2. Determinations of the amount of the primary electron acceptors associated with Photosystem II point to a 2- to 3-fold larger electron acceptor pool in chloroplasts of young Su/su plants than in chloroplasts of old Su/su plants and of various green leaves, including those from green tobacco. This finding agrees with recently published data on the size of the photosynthetic unit in tobacco mutants and normal green plants.

3. The different fluorescence characteristics of all four types of chloroplasts under study are discussed on the basis of their structure and their activity in photosynthetic O₂ evolution.