Fluorescent Methods to Study DNA,RNA, Proteins and Cytoplasmic Membrane Polarization in the Pentachlorophenol-Mineralizing Bacterium <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. UG30 During Nutrient Starvation in Water

The effect of sodium pentachlorophenolate (NaPCP) exposure on the nutrient-starved pentachlorophenol (PCP)-mineralizing bacterium Sphingomonas sp. UG30 was assessed using fluorescent methods to measure DNA, RNA, total cellular protein, and cytoplasmic membrane proteins. UG30 cells were inoculated into sterilized Speed River (Guelph, ON, Canada) water samples in the presence of 50, 100, and 250 ppm NaPCP. No marked changes were observed in the total cellular DNA, RNA or protein levels over 90 d, indicating the macromolecular composition of UG30 was not affected by both nutrient limitation and NaPCP. Total cell counts as determined by DAPI staining also did not change over 90 d. Over the same period, viable counts decreased with increasing concentrations of NaPCP. At 250 ppm NaPCP, viable cell counts decreased over 6 orders of magnitude after 1 hr exposure. Cell numbers partially recovered once NaPCP was degraded. The UG30 cytoplasmic membrane polarization ratio also decreased after NaPCP was depleted. The decreased polarization value at the end of the study period suggested the UG30 membrane was more fluid and that this increase in fluidity was due to nutrient starvation effects rather than exposure to NaPCP. These results indicated that UG30 is a robust organism that is able to degrade NaPCP even under adverse conditions and fluorescent methods are useful for determining macromolecular concentrations and cytoplasmic membrane polarization values.     

Stress-Induced Alteration of Chlorophyll Fluorescence Polarization and Spectrum in Leaves of <Emphasis Type="Italic">Alocasia macrorrhiza</Emphasis> L. Schott

The value of intrinsic chlorophyll fluorescence polarization, and the intensity in emission spectrum were investigated in leaf segments of Alocasia macrorrhiza under several stress conditions including different temperatures (25–50°C), various concentrations of NaCl (0–250 mM), methyl viologen (MV, 0–25 μM), SDS (0–1.0%) and NaHSO₃ (0–80 μM). Fluorescence emission spectrum of leaves at wavelength regions of 500–800 nm was monitored by excitation at 436 nm. The value of fluorescence polarization (P value), as result of energy transfer and mutual orientation between chlorophyll molecules, was determined by excitation at 436 nm and emission at 685 nm. The results showed that elevated temperature and concentrations of salt (NaCl), photooxidant (MV), surfactant (SDS) and simulated SO₂ (NaHSO₃) treatments all induced a reduction of fluorescence polarization to various degrees. However, alteration of the fluorescence spectrum and emission intensity of F₆₈₅ and F₇₃₁ depended on the individual treatment. Increase in temperature and concentration of NaHSO₃ enhanced fluorescence intensity mainly at F₆₈₅, while an increase in MV concentration led to a decrease at both F₆₈₅ and F₇₃₁. On the contrary, NaCl and SDS did not cause remarkable change in fluorescence spectrum. Among different treatments, the negative correlation between polarization and fluorescence intensity was found with NaHSO₃ treatments only. We concluded that P value being measured with intrinsic chlorophyll fluorescence as probe in leaves is a susceptible indicator responding to changes in environmental conditions. The alteration of P value and fluorescence intensity might not always be shown a functional relation pattern. The possible reasons of differed response to various treatments were discussed.     

DNA Vector Polyethyleneimine Affects Cell pH and Membrane Potential: A Time-Resolved Fluorescence Microscopy Study

Polyethyleneimine (PEI) is one of the very efficient nonviral vectors being developed and tested for artificial gene transfer into target cells. One of its serious limitations is the significant cytotoxicity of the large amounts of free PEI in the mixtures of DNA and PEI used for transfection. To further investigate the cellular effects of free PEI, we have analyzed the PEI-induced alterations of various cell parameters such as membrane heterogeneity and fluidity, cytoplasmic pH, and plasma membrane potential in a variety of cells such as Swiss 3T3 fibroblast, Chinese hamster ovary, insect cells SF9, plant cell line BY2, and Saccharomyces cerevisae. Fluorescence probes such as Nile red, SNARF-1, and cyanine dye DiSC₂(3), coupled with the technique of picosecond time-resolved fluorescence microscopy, were used in estimating the above-mentioned cell parameters. It was found that the cell membranes were largely unperturbed by PEI. However, the cytoplasmic pH showed an increase of 0.1–0.4 units when the cells were treated with PEI. The plasma membrane potential was found to be depolarized in S. cerevisae and Swiss 3T3 cells. These results suggest that the cytotoxic effects of PEI may partly originate from inhibition of regulation of cytoplasmic pH and plasma membrane potential. Further, it is proposed that the resultant cell alterations favors the transfection process.     

Does membrane lipid profile explain chilling sensitivity and membrane lipid phase transition of spermatozoa and oocytes?

Ram, fowl and bee spermatozoa, and oocytes of cows and zebrafish were used to study lipid membrane profiles, chilling sensitivity and lipid-phase transitions. The integrity of the membranes was determined by carboxyfluorescein diacetate (CFDA) staining following exposure for 15 minutes to low temperatures. Ram and fowl spermatozoa showed different degrees of loss of membrane integrity. Surprisingly, bee spermatozoa did not show any sensitivity to chilling, and their membranes remained intact down to 0 degree C. In bovine oocytes (at the GV stage) chilling injury was very severe at 16 degree C (membrane integrity decreased by 50%). Lipid phase transition (LPT) and membrane fluidity, which were evaluated by Fourier transform infrared (FTIR) microscopy, and fluorescence polarisation, showed phase transitions at the same temperatures as caused damage (between 30 and 12 degree C). The membrane lipid profiles showed high concentrations of polyunsaturated fatty acids (PUFA) in cold-sensitive ram spermatozoa and zebrafish oocytes, but the ratio between PUFA and saturated fatty acids was highest in cold-resistant bee spermatozoa and lowest in cold-sensitive bovine oocytes. These results suggest a close relationship among cold susceptibility, lipid phase transition and lipids profile in animal gametes.     

Polarization of the Fluorescence Excited in the P- and R Branches of the Vibronic Lines of Jet-Cooled Complex Molecules

A model is suggested to calculate the degree of polarization of the fluorescence of jet-cooled polyatomic molecules excited within the P- and R branches of the rotational contour of the vibronic line by light not providing resolution of the rotational structure. These dependences are calculated and compared with experimental data for molecules belonging to different types of asymmetric tops with different intramolecular orientation of the fluorescence transition dipole moment: anthracene; anthracene + Ar; 1,4-diaminoanthraquinone; indole; tetracene, and triphendioxazine.     

Anomalous fluorescence properties of 4-N,N-dimethylaminobenzoic acid in polar solvents

4-N,N-Dimethylaminobenzoic acid exhibits anomalous fluorescence in polar and hydrogen-bonding solvents. The fluorescence spectra and kinetics suggest that this arises due to the formation of a ground-state dimer or higher polymer. Preliminary measurements in hexane containing small amounts of polar acetonitrile do not rule out the possibility of exciplex formation also occurring.     

Study of plasma membrane heterogeneity using a phosphatidylcholine derivative of 1,6-diphenyl-1,3,5-hexatriene [2-(3-(diphenylhexatriene)propanoyl)-3-palmitoyl-l-α-phosphatidylcholine]

The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) and DPH derivatives have been used to characterize structural and physicochemical properties of specific membrane domains. Steady-state and fluorescence decay measurements of three probes, DPH (1,6-diphenyl-1,3,5-hexatriene), TMA-DPH [1-(4-trimethyl-ammonium-phenyl)-6-phenyl-1,3,5-hexatriene], and a phosphatidylcholine derivative of DPH, DPH-pPC [2-(3-(diphenylhexatriene)propanoyl)-3-pamitoyl-L--phosphatidyl choline], have been performed in erythrocyte membranes and in lymphocyte plasma membranes. The steady-state fluorescence polarization of the three probes showed a similar trend in both membranes. In fact either in erythrocyte or in lymphocyte plasma membranes the fluorescence polarization values of DPH-pPC and TMA-DPH were similar, but significantly higher with respect to DPH. A better characterization of erythrocyte and lymphocyte plasma membranes was possible by using fluorescence decay measurements. The data suggest the possible use of different DPH derivatives to characterize specific domains in biological membranes.     

Dynamics in Diether Lipid Bilayers and Interdigitated Bilayer Structures Studied by Time-Resolved Emission Spectra, Decay Time and Anisotropy Profiles

We present a comparative fluorescence spectroscopic investigation of diacyl and diether phosphatidylcholine vesicles using different probes with well-defined localization within either the hydrophilic headgroup region or the hydrophobic part of the bilayer. Time-resolved emission spectra have been used to characterize the solvent relaxation behavior in both symmetric and asymmetric diether and diacyl phosphatidylcholines. It is shown that time-resolved emission spectra of Prodan (6-propionyl-2-(dimethylamino)-naphthalene) and its long-alkyl chain derivative Patman (6-palmitoyl-2-[[trimethylammoniumethyl]methylamino]-naphthalene chloride) are a sensitive tool for the detection of differences in the micropolarities and viscosities at the hydrophobic/hydrophilic membrane interface of diether and diacyl lipids, respectively. Moreover, a new approach for the detection of interdigitated bilayers is discussed. It relies on the construction of anisotropy and decay time profiles for the set of n-anthroyloxy fatty acids and is compared with an older fluorescence assay based on intensity measurements only. The shape of plots of the fluorescence steady-state anisotropy versus the position of the chromophore (anthracene-9-carboxylic acid) combined with fluorescence lifetime measurements can be used to differentiate among non-fully, and mixed interdigitated gel phase structures and to predict structures for new lipid species.     

运用偏振荧光的手段研究氧化胁迫对小麦叶绿体荧光的影响

采集抽穗期小麦旗叶,采用1mmol.L-1 H2O2、干旱、黑暗处理24h诱导产生氧化损伤模型,然后运用偏振荧光的手段检测了叶绿体的荧光发射谱和荧光激发谱,结果发现,无论选择436nm激发叶绿素a(Chla)分子,或固定475nm激发叶绿素b(Chlb)分子,氧化胁迫后光系统Ⅱ反应中心P680与光系统Ⅰ反应中心P700的荧光发射峰峰面积比值A684/A720呈上升趋势;通过比较偏振荧光激发谱上E436/E475和E475/E600比值,发现随着氧化胁迫的进行,Chla对于反应中心能量传递的相对贡献大于Chlb;此外,类胡萝卜素向Chlb能量传递效率在各个偏振方向上均有所提高;通过计算偏振度及粘度,发现氧化胁迫处理促使680nm处荧光偏振度提高,内囊体膜微环境粘度增加。上述结果为研究氧化胁迫提供了一种简单、易行的方法。     

Membrane fluidity altering and enzyme inactivating in sarcoma 180 cells post the exposure to sonoactivated hematoporphyrin in vitro

Sonodynamic therapy (SDT) is a novel tumor therapy method. We investigated membrane fluidity, activity of the enzymes and membrane morphology in vitro post hematoporphyrin-SDT treatment. Furthermore, the potential mechanisms behind the changes in membrane fluidity and enzymic activity were discussed. Tumor cells were exposed to ultrasound at 1.75 MHz for up to 3 min in the presence and absence of hematoporphyrin. Fluorescence polarization, contents of Malonaldehyde, and levels of free fatty acid were assessed. Activity of enzymes was checked by the plumbic nitrate detection method. For the morphologic study, a scanning electron microscope was used to observe the cellular surface. Ultrasonically induced cell damage increased in the presence of HPD (from 15% to 24%). Compared with ultrasound treatment alone, the fluidity decreased from 5.037 to 3.908, malonaldehyde content and free fatty acid level increased from 0.743 nmol/mL to 0.979 nmol/mL and from 237.180 μmol/L to 730.769 μmol/L, respectively, post ultrasound combined with HPD treatment. Inactivity of adenylate cyclase and guanylate cyclase and significant deformation of the cellular surface were also observed post SDT treatment. Our results suggested that alterations in membrane modality and lipid composition played important roles in SDT-mediated inhibition of tumor growth, even inducing tumor cell death, which might be attributed to a sono-chemical activation mechanism.     

Linearly-polarized Tm-doped double-clad fiber laser

We report a linearly polarized Tm doped fiber laser. The fiber laser was set up by using a piece of polarization maintaining Tm doped double clad fiber of 5 m length as gain medium and a polarization beam splitter as a polarization selector. The fiber laser was pumped by a fiber pigtailed laser diode working at 790 nm with a maximum output power of 90 W. The linearly polarized Tm laser operated at wavelength around 2030 nm. A maximum output power up to 21.9 W was achieved when the pump power was 63.27 W with a threshold of 11.92 W, a slope efficiency of about 43.7%, and a polarization extinction ratio of 92.7% (11.37 dB). In addition to the blue fluorescence, we also observed the violet fluorescence under high pump power level. The up-conversion fluorescence was considered to be attributed to the ¹ G ₄ → ³ H ₆, and ¹ D ₂ → ³ F ₄ transitions of Tm ions, respectively.     

Laser-induced fluorescence measurements and modeling of absolute CH concentrations in strained laminar methane/air diffusion flames

We have applied linear laser-induced fluorescence to obtain spatially resolved profiles of CH radicals in laminar methane/air and methane/nitric oxide/air counterflow diffusion flames at atmospheric pressure. Excitation and detection of transitions in the A–X band and calibrating the optical detection efficiency via Rayleigh scattering allowed the determination of absolute radical concentrations. Flames at strain rates from 59 to 269 s⁻¹ were studied to characterize the strain rate dependence of the CH concentration. The work shows that CH concentrations increase with increasing strain rate. Comparisons have been made with predicted CH levels obtained using two different chemical kinetic mechanisms (Lindstedt et al. and GRI-Mech. 3.0). Computed concentrations are shown to be in good agreement with experimental data. It was furthermore found that the addition of up to 600 ppm NO to the fuel did not have a measurable effect on the CH radical concentration. This is also in agreement with predictions from both mechanisms. The current work has shown that measurements of absolute CH radical concentrations are possible in non-premixed flames without the need for spatial temperature or quenching corrections.     

Locating of Nucleic Acid Intercalators in Yeast Cells by Image Analysis Combined Fluorescence Microscopy

Intracellular distribution in the intact (not fixed) Saccharomyces cerevisiae cells of the nucleic acid intercalators (NAI) was studied using fluorescence microscopy combined with computer image analysis (ImageJ software, NIH, USA). Three NAI—the anthracycline anticancer drug doxorubicin (DR) along with the nucleic acid dyes ethidium (E) and 4′,6-diamidino-2-phenylindole (DAPI)—were used. Staining pattern and ImageJ quantitative analysis data provided evidence that all three NAI were located in the nuclei and in the mitochondria. DR and E, in contrast to DAPI, may be bound to not only DNA, but to the mitochondrial membranes as well. Experiments on the combined application of DR+DAPI and E+DAPI have shown potential competition of DAPI with DR and E for binding sites in the nuclear and mitochondrial DNA. With the approach presented herein, the yeast cells of S. cerevisiae can be used as a model for locating intracellular sites of the fluorescing nucleic acid intercalators. This model may be of help in designing new DNA-targeted drugs and in preliminary studies of their interaction with eukaryotic cells.     

Optical,nanostructural, and biophysical properties of Zn-induced changes in human erythrocyte membranes

We studied changes in the surface of erythrocyte membranes exposed to the action of zinc sulfate in the concentration range of 0.1–2.0 mM/l using methods of light scattering, spectrofluorimetry, and atomic force microscopy. Using the spectrofluorimetry method, we revealed a dose-dependent increase in the fluorescence intensity of a fluorescamine probe incorporated into erythrocyte membranes modified by zinc ions, which is indicative of an increase in the level of NH₂ groups on the cell surface. Using atomic force microscopy, we revealed changes in the surface topography of erythrocyte membranes exposed to the action of zinc sulfate in the concentration range of 0.1–2.0 mM/l. By performing a correlation analysis, we revealed that the correlation length of the autocorrelation function of the erythrocyte surface irregularity profile directly related to the fluorescence intensity of fluorescamine incorporated into erythrocyte membranes (r = 0.9, p < 0.05) modified by zinc ions. We showed that, in the zinc sulfate concentration range of 0.1–2.0 mM/l, zinc oxides form in erythrocyte membranes, which is confirmed by the appearance of an absorption band at 330–340 nm and by an increase in the light scattering. At more considerable concentrations, we identified absorption bands characteristic of zinc protein complexes in erythrocyte membranes. A considerable decrease in the elongation of the scattering indicatrix of erythrocyte membranes caused by luminescence correlates with the content of zinc proteins. Polarization measurements confirm the enhancement of the aggregation of protein complexes observed by the atomic force microscopy method. The proposed complex approach can be used in studies on the action of various abiotic factors on biological cells.     

The changing biochemical composition and organisation of the murine oocyte and early embryo as revealed by Raman spectroscopic mapping

Despite an exponential uptake in recent years of assisted reproductive techniques, such as in vitro fertilisation, much is still not fully understood about the biochemical modifications that take place during the development and maturation of the egg and embryo. As such, in order to improve the efficiency of these techniques, furthering our understanding of the processes that underpin oocyte and embryo development is necessary. Raman spectroscopic mapping as a technique enables the investigation of biochemical variation within intact cells without the need for labelling. Here, Raman maps of fixed immature and mature oocytes along with early stage embryos were collected using 785 nm excitation and a step size of 2 µm. The results were analysed using both univariate and multivariate techniques. It was found that significant macromolecular accumulation took place during oocyte maturation, while a decrease in total lipid content consistent with the formation of new cellular membranes is observed upon embryo cleavage. Furthermore, an observed asymmetrical localisation of macromolecules in the mature oocyte may indicate the existence of cytoplasmic polarisation, a phenomenon that has been observed in the eggs of lower organisms. As such, these results indicate that Raman spectroscopic mapping may present an alternative analytical tool for investigating the biochemistry of egg and embryo development. In particular, these results indicate that temporal Raman analysis may help to reveal the existence of cytoplasmic polarisation in the murine egg. Copyright © 2011 John Wiley & Sons, Ltd.     

Steady-State and Time-Resolved Fluorescence Polarization Behavior of Acenaphthene

Steady-state and time-resolved fluorescence polarization studies have been carried out on acenaphthene (ACE) in low-temperature glass solutions and at room temperature. In the low-temperature glass the fluorescence polarization values vary considerably with both emission and excitation wavelength. There is a time dependence (on the nanosecond time scale) of the fluorescence anisotropy, r(t), at 77 K, which has a strong dependence upon the excitation and emission wavelengths. Under these conditions, the time-dependent decay of the anisotropy is not attributable to chromophoric motion. The observations are consistent with emission from two closely lying and interconverting excited states. Rate constants for the photophysical processes involved have been determined by fitting the data using a model proposed by Fleming et. al. The results are discussed with particular reference to the care required in using dynamic fluorescence polarization measurements to determine energy transfer rates in systems containing this chromophore.     

基于偏振技术的藻类水体散射光中叶绿素荧光分离方法研究

浮游藻类细胞内的叶绿素分子吸收光能后,会以释放能量的形式发射叶绿素荧光。水中颗粒物的弹性散射光是部分偏振光,而叶绿素荧光则是非偏振光,因此利用这一特性可以从总散射光谱中分离出荧光,从而达到反演叶绿素浓度的目的。但对于近岸等复杂水体,该方法能否适用并不清楚,基于此,实验通过测量分别分析了不同无机颗粒物浓度和叶绿素浓度对偏振分离方法的影响。结果表明,对含不同浓度无机颗粒物的藻类水体,分离出的荧光峰会随浓度增加有降低趋势,但是当颗粒物浓度达到300 mg·L-1时,反演结果仍然可靠。对含不同浓度叶绿素的藻类水体,叶绿素浓度越高,偏振方法分离荧光的效果越好,对低浓度正常水体,仍能识别。实验证明了利用偏振方法分离叶绿素荧光对复杂水体仍然有效,对进一步遥感探测近岸水体的叶绿素浓度具有重要意义。     

Polarity dependence of EPR parameters for TOAC and MTSSL spin labels: correlation with DOXYL spin labels for membrane studies

TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) is a nitroxyl amino acid that can be incorporated in the backbone of peptides. DOXYL (4,4-dimethyl-oxazolidine-1-oxyl) is a nitroxyl ring that can be attached rigidly at specific C-atom positions in the acyl chains of phospholipids. Spin-labelled phosphatidylcholines of the DOXYL type have been used previously to establish the transmembrane polarity profile in biological lipid bilayers [D. Marsh, Polarity and permeation profiles in lipid membranes, Proc. Natl. Acad. Sci. USA 87 (2001) 7777-7782]. Here, we determine the polarity dependence of the isotropic (14)N-hyperfine couplings, a(o)(N), and g-values, g(o), in a wide range of protic and aprotic media, for a TOAC-containing dipeptide (Fmoc-TOAC-Aib-OMe) and for a DOXYL-containing fatty acid (12-DOXYL-stearic acid). The correlation between datasets for TOAC and DOXYL nitroxides in the various solvents is used to establish the polarity profile for isotropic hyperfine couplings of TOAC in a transmembrane peptide. This calibration can be used to determine the location of TOAC at selected residue positions in a transmembrane or surface-active peptide. A similar calibration procedure is also applied to a(o)(N) and g(o) for the pyrroline methanethiosulphonate nitroxide (MTSSL) that is used in site-directed spin-labelling studies of membrane proteins.     

Spectral-Kinetic Characteristics of Pyrene Fluorescence in Model Systems and Membranes of Etioplasts

The decay kinetics parameters of monomer and excimer fluorescence of pyrene in artificial galactolipid protein-free micelles and membranes of prolamellar bodies (PLB) of etioplasts have been determined. A complex law of probe fluorescence decay in artificial and native membranes has been discovered. It has been found that the addition of protochlorophyllide (Pd) to lipid micelles led to a considerable reduction in the lifetime of the long-lived component of the fluorescence-decay kinetics of the monomer form of pyrene (₂) and to the appearance of luminescence in the region of 640 nm in the stationary spectra of fluorescence at excitation of the pyrene molecules present in the bilayer. In native membranes of etioplasts, the monomer duration ₂ did not depend on the pigment content and the Pd fluorescence upon excitation through pyrene was absent. The membranes of etioplasts and the pigment-containing artificial micelles did not differ in the ₂ value of the excimer. They only differed in the contribution of the long-lived component to the total fluorescence (low in native membranes). The pigment-protein interaction in the etioplast membranes and the absence from them of pigment directly associated with lipids are discussed.     

In situ studies of microbial inactivation during high pressure processing

High pressure processing (HPP) has been shown to reduce microbial concentration in foods. The mechanisms of microbial inactivation by HPP have been associated with damage to cell membranes. The real-time response of bacteria to HPP was measured to elucidate the mechanisms of inactivation, which can aid in designing more effective processes. Different pressure cycling conditions were used to expose Enterobacter aerogenes cells to HPP. Propidium iodide (PI) was used as a probe, which fluoresces after penetrating cells with damaged membranes and binding with nucleic acids. A HPP vessel with sapphire windows was used for measuring fluorescence in situ. Membrane damage was detected during pressurization and hold time, but not during depressurization. The drop in fluorescence was larger than expected after pressure cycles at higher pressure and longer times. This indicated possible reversible disassociation of ribosomes resulting in additional binding of PI to exposed RNA under pressure and its release after depressurization.