A combined micro-resonance Raman and absorption set-up enabling in vivo studies under varying physiological conditions: The nerve globin in the nerve cord of Aphrodite aculeata

We hereby report on the design of a set-up combining micro-resonance Raman and absorption spectroscopy with a microfluidic system. The set-up enabled us to study the nerve globin of Aphrodite aculeata in the functional isolated nerve cord under varying physiological conditions for extended periods of time. The oxygenation cycle of the organism was triggered by utilizing the microfluidic system that allowed for a fast switch between aerobic and anaerobic conditions. The nerve globin was found to very easily shift from a penta-coordinated high spin ferrous form to the oxy state upon a change from anaerobic to aerobic conditions. The observed fast reaction to varying O₂ concentrations supports an oxygen-carrying and/or -storing function of the nerve globin. In addition, by combining resonance Raman and absorption spectroscopy, the physiological response could be distinguished from light-induced effects.     

Tyrosinase inactivation in its action on dopa

Under aerobic or anaerobic conditions, tyrosinase undergoes a process of irreversible inactivation induced by its physiological substrate l-dopa. Under aerobic conditions, this inactivation occurs through a process of suicide inactivation involving the form oxy-tyrosinase. Under anaerobic conditions, both the met- and deoxy-tyrosinase forms undergo irreversible inactivation. Suicide inactivation in aerobic conditions is slower than the irreversible inactivation under anaerobic conditions. The enzyme has less affinity for the isomer d-dopa than for l-dopa but the velocity of inactivation is the same. We propose mechanisms to explain these processes.     

Raman spectroscopy of nerve fibers. A study of membrane lipids under steady state conditions.

The molecular structures of different nerve fibers kept in good physiological conditions were studied by laser Raman spectroscopy. For myelinated nerves like the rat sciatic nerve, the Raman spectrum is dominated by bands due to the lipid component of the myelin sheath. The temperature dependence of these bands does not reveal any thermotropic phase transition between 0 and 40 degrees C. There is, however, with temperature, a linear increase in the intermolecular disorder that is accompanied by an increase in the number of gauche bonds of the phospholipid acyl chains. For unmyelinated nerves such as the lobster leg nerve, the C-H stretching region of the Raman spectrum is covered by bands arising from the protein component of the axoplasm. However, for the garfish olfactory nerve that has a high density of excitable membranes, phospholipid bands are observed and can be used as intrinsic structural probes of the excitable membranes. The relative intensity of these bands is also temperature dependent.     

Anaerobic fumarate transport in Escherichia coli by an fnr-dependent dicarboxylate uptake system which is different from the aerobic dicarboxylate uptake system.

Escherichia coli grown anaerobically with fumarate as electron acceptor is able to take up C4-dicarboxylates by a specific transport system. The system differs in all tested parameters from the known aerobic C4-dicarboxylate transporter. The anaerobic transport system shows higher transport rates (95 mumol/g [dry weight] per min versus 30 mumol/g/min) and higher Kms (400 versus 30 microM) for fumarate than for the aerobic system. Mutants lacking the aerobic dicarboxylate uptake system are able to grow anaerobically at the expense of fumarate respiration and transport dicarboxylates with wild-type rates after anaerobic but not after aerobic growth. Transport by the anaerobic system is stimulated by preloading the bacteria with dicarboxylates. The anaerobic transport system catalyzes homologous and heterologous antiport of dicarboxylates, whereas the aerobic system operates only in the unidirectional mode. The anaerobic antiport is measurable only in anaerobically grown bacteria with fnr+ backgrounds. Additionally, the system is inhibited by incubation of resting bacteria with physiological electron acceptors such as O2, nitrate, dimethyl sulfoxide, and fumarate. The inhibition is reversed by the presence of reducing agents. It is suggested that the physiological role of the system is a fumarate/succinate antiport under conditions of fumarate respiration.     

Resonance Raman study on photoreduction of cytochrome c oxidase: distinction of cytochromes a and a3 in the intermediate oxidation states

Occurrence of photoreduction of bovine cytochrome c oxidase was confirmed with the difference absorption spectra and oxygen consumption measurements for the enzyme irradiated with laser light at 406.7, 441.6, and 590 nm. The resonance Raman spectra were obtained under the same experimental conditions as those adopted for the measurements of oxygen consumption and difference absorption spectra. The photoreduction was more effective upon irradiation at shorter wavelengths and was irreversible under anaerobic conditions. However, upon aeration into the cell, the original oxidized form was restored. It was found that aerobic laser irradiation produces a photo steady state of the catalytic dioxygen reduction and that the Raman scattering from this photo steady state probes cytochrome a2+ and cytochrome a3(3)+ separately upon excitations at 441.6 and 406.7 nm, respectively. The enzyme was apparently protected from the photoreduction in the spinning cell with the spinning speed between 1 and 1500 rpm. These results were explained satisfactorily with the reported rate constant for the electron transfer from cytochrome a to cytochrome a3 (0.58 s-1) and a comparable photoreduction rate of cytochrome a. The anaerobic photoreduction did give Raman lines at 1666 and 214 cm-1, which are characteristic of the ferrous high-spin cytochrome a3(2)+, but they were absent under aerobic photoreduction. The formyl CH = O stretching mode of the a3 heme was observed at 1671 cm-1 for a2+a3(2)+CO but at 1664 cm-1 for a2+a3(2)+CN-, indicating that the CH = O stretching frequency reflects the pi back-donation to the axial ligand similar to the oxidation state marker line (v4).     

光镊拉曼光谱分析酿酒活性干酵母的活化与生长

应用光镊拉曼光谱新技术(LTRS)对酿酒活性干酵母复水活化与生长进行动态观察, 探索从分子光谱角度窥视胞内糖类、核酸、蛋白等生物大分子的变化过程, 及葡萄糖消耗和乙醇生成的动态过程。结果显示, 酿酒活性干酵母复水活化后, 第6小时和9小时, 即酵母对数生长中期及乙醇产生前期, 是调控酵母细胞生理变化的2个重要的时间点。核酸类物质在细胞活化后迅速增加, RNA在第6小时达到最大值; 而蛋白质和脂类物质从第6小时开始快速增加, 在第9小时达 到最大值, 而后呈下降趋势; 胞内乙醇则是在9 h开始出现, 在9     

Spectral analyses of absorption changes associated with nerve excitation in dye-stained crab nerve

Spectral characteristics of absorption changes associated with nerve excitation were studied with crab nerves stained with a homologous series of dyes, merocyanine-rhodanines and rhodanine oxonols. In these classes of dyes, the absorption changes which followed approximately the same time course as that of the action potential (fast responses) depended in a similar fashion on the wavelength and polarization of the incident light. In order to interpret those commonly observed dependencies, a mode of reorientation of the absorption oscillators of the dye molecules in the membrane matrix during nerve excitation was proposed. In addition to the fast changes mentioned above, slow responses which developed during and after the action potential were commonly observed with oxonols. The spectra of the slow changes differed from those of the fast ones, indicating a distinct mechanism on the response production. A possible mechanism of the production of fast responses was also discussed based on the proposed mode of reorientation of the absorption oscillators.     

Oxygen and temperature-dependent structural and redox changes in a novel cytochrome c(4) from the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina

A novel cytochrome c₄, the first of this type in purple phototrophic bacteria has been discovered in Thiocapsa roseopersicina. The fact that cytochrome c₄ has been found in an anaerobic organism puts in question the up hereto suggested role of cytochromes c₄ in the aerobic respiratory metabolism. The structure of cytochrome c₄ was studied under both aerobic and anaerobic conditions, using differential scanning calorimetry and a combination of redox potentiostatic measurements with CD and UV-Vis absorption techniques. Cytochrome c₄ maintained its functional capability at high temperature (60 °C) if it was kept under anaerobic conditions. With increasing temperature under aerobic conditions, however, there are dramatic conformational changes in the protein and coordination changes on the iron side. Presumably oxygen binds to the iron at the position left vacant by the methionine and facilitates conformational changes with low reversibility.     

Carbohydrate metabolism in the isolated perfused rat kidney

1. Anaerobic formation of lactate from glucose by isolated perfused rat kidney (411mumol/h per g dry wt.) was three times as fast as in aerobic conditions (138mumol/h per g). 2. In aerobic or in anaerobic conditions, the ratio of lactate production to glucose utilization was about 2. 3. Starvation or acidosis caused a decline of about 30% in the rate of aerobic glycolysis. 4. The rate of formation of glucose from lactate by perfused kidney from a well-fed rat, in the presence of 5mm-acetoacetate (83mumol/h per g dry wt.), was of the same order as the rate of aerobic glycolysis. 5. During perfusion with physiological concentrations of glucose (5mm) and lactate (2mm) there were negligible changes in the concentration of either substrate. 6. Comparison of kidneys perfused with lactate, from well-fed or starved rats, showed no major differences in contents of intermediates of gluconeogenesis. 7. The tissue concentrations of hexose monophosphates and C(3) phosphorylated glycolytic intermediates (except triose phosphate) were decreased in anaerobic conditions. 8. Aerobic metabolism of fructose by perfused kidney was rapid: the rate of glucose formation was 726mumol/h per g dry wt. and of lactate formation 168mumol/h per g (dry wt.). Glycerol and d-glyceraldehyde were also released into the medium. 9. Aerobically, fructose generated high concentrations of glycolytic intermediates. 10. Anaerobic production of lactate from fructose (74mumol/h per g dry wt.) was slower than the aerobic rate. 11. In both anaerobic and aerobic conditions the ratio [lactate]/[pyruvate] in kidney or medium was lower during perfusion with fructose than with glucose. 12. These results are discussed in terms of the regulation of renal carbohydrate metabolism.     

Pyrimidine dimer excision repair of DNA in Bacteroides fragilis wild-type and mitomycin C-sensitive/UV-sensitive mutants

An enzyme preparation purified from Micrococcus luteus was shown to be specific for UV-induced pyrimidine dimers and was suitable for the detection of DNA excision repair systems. The wild-type Bacteroides fragilis Bf-2 strain and a mitomycin C-sensitive mutant (MTC25) had constitutive dimer excision systems which functioned efficiently under anaerobic and aerobic conditions. A UV-sensitive mutant (UVS9) had markedly reduced levels of the constitutive dimer excision systems under anaerobic and aerobic conditions. Since liquid holding recovery under aerobic conditions was inhibited by chloramphenicol whereas the final level of excision repair in B. fragilis Bf-2 was not affected, it is concluded that pyrimidine dimer removal is not the process responsible for increased physiological aerobic liquid holding recovery.     

Induced cooperation between marine nitrifiers and anaerobic ammonium-oxidizing bacteria by incremental exposure to oxygen

In oxygen-limited marine ecosystems cooperation between marine nitrifiers and anaerobic ammonium-oxidizing (anammox) bacteria is of importance to nitrogen cycling. Strong evidence for cooperation between anammox bacteria and nitrifiers has been provided by environmental studies but little is known about the development of such communities, the effects of environmental parameters and the physiological traits of their constituents. In this study, a marine laboratory model system was developed. Cooperation between marine nitrifiers and anammox bacteria was induced by incremental exposure of a marine anammox community dominated by Scalindua species to oxygen in a bioreactor set-up under high ammonium (40 mM influent) conditions. Changes in the activities of the relevant functional groups (anammox bacteria, aerobic ammonia oxidizers and nitrite oxidizers) were monitored by batch tests. Changes in community composition were followed by Fluorescence in situ Hybridization (FISH) and by amplification and sequencing of 16S rRNA and amoA genes. A co-culture of Scalindua sp., an aerobic ammonia-oxidizing Nitrosomonas-like species, and an aerobic (most likely Nitrospira sp.) nitrite oxidizer was obtained. Aerobic ammonia oxidizers became active immediately upon exposure to oxygen and their numbers increased 60-fold. Crenarchaea closely related to the ammonia-oxidizer Candidatus 'Nitrosopumilus maritimus' were detected in very low numbers and their contribution to nitrification was assumed negligible. Activity of anammox bacteria was not inhibited by the increased oxygen availability. The developed marine model system proved an effective tool to study the interactions between marine anammox bacteria and nitrifiers and their responses to changes in environmentally relevant conditions.     

Parallelized shifted‐excitation Raman difference spectroscopy for fluorescence rejection in a temporary varying system

A fluorescence background is one of the common interference factors of the Raman spectroscopic analysis in the biology field. Shifted‐excitation Raman difference spectroscopy (SERDS), in which a slow (typically 1 Hz) modulation to excitation wavelength is coupled with a sequential acquisition of alternating shifted‐excitation spectra, has been used to separate Raman scattering from excitation‐shift insensitive background. This sequential method is susceptible to spectral change and thus is limited only to stable samples. We incorporated a fast laser modulation (200 Hz) and a mechanical streak camera into SERDS to effectively parallelize the SERDS measurement in a single exposure. The developed system expands the scope of SERDS to include temporary varying system. The proof of concept is demonstrated using highly fluorescent samples, including living algae. Quantitative performance in fluorescence rejection and the robustness of the method to the dynamic spectral change during the measurement are manifested.      

An aerobic fixed-phase biofilm reactor system for the degradation of the low-molecular weight aromatic compounds occurring in the effluents of anaerobic digestors treating olive mill wastewaters

An aerobic co-culture, prepared by combining Ralstonia sp. LD35 and Pseudomonas putida DSM1868, was recently found to be capable of extensively degrading many of the hydroxylated and/or methoxylated benzoic, phenylacetic and 3-phenyl-2-propenoic acids occurring in the olive mill wastewaters (OMWs). In the perspective of developing a biotechnological process for the degradation of low-molecular weight (MW) aromatic compounds occurring in the effluents of anaerobic digestors treating OMWs, the capability of this bacterial co-culture of biodegrading a synthetic mix of the above mentioned compounds and the aromatic compounds of an anaerobic OMW-treatment plant effluent in the physiological state of immobilised cells was investigated. Two aerobic fixed-bed biofilm reactors were developed by immobilising the co-culture cells on Manville silica beads and on polyurethane foam cubes. Both supports were found to give rise to a microbiologically stable and biologically active biofilm. The two biofilm reactors were found to be similarly capable of rapidly and completely biodegrading the components of a synthetic mix of nine monocyclic aromatic acids typically present in OMWs and the low-MW aromatic compounds occurring in the anaerobic effluent in batch conditions. However, in the same conditions, the silica bead-packed reactor was found to be more effective in the removal of high-MW phenolic compounds from the anaerobic effluent with respect to the polyurethane cube-packed reactor. These results are encouraging in the perspective of using the co-culture as immobilized cells for developing a continuous biotechnological process for the post-treatment of effluents with low-MW aromatic compounds produced by anaerobic digestors treating OMWs.     

Changes in Shewanella putrefaciens CN32 Membrane Stability upon Growth in the Presence of Soluble Mn(II), V(IV), and U(VI)

In natural reducing environments, such as anoxic sediments and soils, bacteria may be exposed to high concentrations of soluble transition metals. The aim of this study was to identify physiological and biochemical adaptations of Shewanella putrefaciens CN32 membranes to soluble Mn(II), V(IV), and U(VI). We assessed responses of CN32 to these metals, in aerobic and anaerobic cultures, by means of membrane fluidity and fatty acid composition assays. During aerobic growth, all metals had a stabilizing effect on fluidity, while under anoxic conditions this was only observed for bacteria treated with U(VI). Membrane gel-to-fluid phase transition temperatures were higher under anaerobic conditions and were not affected by the metal treatments. Fatty acid desaturation demonstrated linear correlation with significant increases in membrane fluidity, despite metal treatments that did not significantly alter fatty acid chemistry. Scanning transmission X-ray microscopy (STXM) and near-edge X-ray absorption fine structure spectroscopy (NEXAFS) at Mn 2p- and V 2p-edges revealed that both Mn(II) and V(IV) were associated with CN32 membranes, with V(IV) associating as VO²⁺ under anoxic conditions only. The results of this study indicate that the bacterial growth environment greatly impacts membrane chemistry and stability, with overall implications for in vitro as well as in situ studies. Supplemental materials are available for this article. Please go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.     

Aerobic and anaerobic swimming speeds of spermatozoa investigated by twin beam laser velocimetry.

The motility of bovine and ovine spermatozoa has been studied under aerobic and anaerobic conditions, using a dual beam laser velocimeter. Cells swimming under aerobic conditions were found to be characterized by a translational swimming speed and a rotation rate that were approximately double those of cells swimming in an anaerobic environment. Both types of spermatozoa have been found to exhibit a sudden coordinated transition between fast and slow swimming states when the available oxygen is exhausted. This transition from aerobic to anaerobic swimming states has also been shown to be reversible. Studies of the duration of aerobic motility using the same apparatus have shown that the cells have a constant motile efficiency over the temperature range 32 degrees-42 degrees C.     

Redox and specific effects of vanadium ions on respiratory enzymes

The effects of vanadium ions on the activities of enzymes of aerobic and anaerobic respiratory chains were investigated in vitro and in situ employing ¹H-, ¹⁴N-, ³¹P- and ⁵¹V- nuclear magnetic resonance spectroscopy, electron paramagnetic resonance spectroscopy and spectrophotometry. Vanadate and vanadyl ions produced either non-specific redox or specific activation or inhibition of respiratory enzymes. The oxidants molybdate and chromate and the reductant dithiothreitol were used to distinguish between non-specific and specific effects of vanadium ions on enzyme activities. The results suggested that components of anaerobic respiratory chains were more susceptible to vanadium ions than those of the aerobic respiratory chain     

一株好氧反硝化菌的分离及特性研究

从土壤中分离得到一株好氧反硝化细菌CY1, 该菌株在厌氧和好氧条件下均具有反硝化能力。硝酸盐氮初始浓度为137.25 mg/L, 30 h内硝酸盐氮去除率分别为99.98%(厌氧)和60.16%(好氧)。通过形态学特征、生理生化特性及16S rDNA同源性比较对菌株CY1进行鉴定, 初步判断CY1为泛养副球菌(Paracoccus pantotrophus)。     

Circular dichroism of the peripheral chlorophylls in photosystem II reaction centers revealed by electrochemical oxidation

Visible absorption spectra and circular dichroism (CD) of the red absorption band of isolated photosystem II reaction centers were measured at room temperature during progressive bleaching by electrochemical oxidation, in comparison with aerobic photochemical destruction, and with anaerobic photooxidation in the presence of the artificial electron acceptor silicomolybdate. Initially, selective bleaching of peripheral chlorophylls absorbing at 672 nm was obtained by electrochemical oxidation at +0.9 V, whereas little selectivity was observed at higher potentials. Illumination in the presence of silicomolybdate did not cause a bleaching but a spectral broadening of the 672-nm band was observed, apparently in response to the oxidation of carotene. The 672-nm absorption band is shown to exhibit a positive CD, which accounts for the 674-nm shoulder in CD spectra at low temperature. The origin of this CD is discussed in view of the observation that all CD disappears with the 680-nm absorption band during aerobic photodestruction.     

Gender modulates the energy cost of muscle contraction in untrained healthy subjects. A P magnetic resonance spectroscopy analysis

The forearm flexor muscles of 56 untrained volunteers (26 women and 30 men) were examined by ³¹P magnetic resonance spectroscopy, during a rest-exercise-recovery protocol, in order to document the impact of gender on muscle energetics. Absolute concentrations of high-energy phosphate compounds, intracellular pH and rates of aerobic and anaerobic ATP production were calculated. An inverse correlation was found between body mass index (BMI) and power output in women but not in men. After correcting for power output and BMI, the measured energy cost of contraction was twice larger for women than for men. This increase was also reflected in larger ATP production from aerobic and anaerobic pathways. This higher energy cost might be explained in part by differences in local muscle mass, a higher impact of fatness, but also by a reduced metabolic efficiency of muscle fibers in untrained women.