Responses of peripheral blood flow to acute hypoxia and hyperoxia as measured by optical microangiography

Oxygen availability is regarded as a critical factor to metabolically regulate systemic blood flow. There is a debate as to how peripheral blood flow (PBF) is affected and modulated during hypoxia and hyperoxia; however in vivo evaluating of functional PBF under oxygen-related physiological perturbation remains challenging. Microscopic observation, the current frequently used imaging modality for PBF characterization often involves the use of exogenous contrast agents, which would inevitably perturb the intrinsic physiologic responses of microcirculation being investigated. In this paper, optical micro-angiography (OMAG) was employed that uses intrinsic optical scattering signals backscattered from blood flows for imaging PBF in skeletal muscle challenged by the alteration of oxygen concentration. By utilizing optical reflectance signals, we demonstrated that OMAG is able to show the response of hemodynamic activities upon acute hypoxia and hyperoxia, including the modulation of macrovascular caliber, microvascular density, and flux regulation within different sized vessels within skeletal muscle in mice in vivo. Our results suggest that OMAG is a promising tool for in vivo monitoring of functional macro- or micro-vascular responses within peripheral vascular beds.     

Stress-induced environmental changes in a single cell as revealed by fluorescence lifetime imaging.

The fluorescence lifetime image of HeLa cells expressing an enhanced green fluorescent protein (EGFP)-fusion protein changes under stress, which allows noninvasive determination of the status of individual cells.     

Cheek cell membrane fluidity measured by fluorescence recovery after photobleaching and steady-state fluorescence anisotropy

Membrane fluidity of human cheek cells was determined using fluorescence recovery after photobleaching (FRAP) and steady-state fluorescence anisotropy. The FRAP data showed that the lateral diffusion coefficient (D) and mobile fraction (%R) of lipid in the plasma membrane of control cells were 2.01×10^–9 cm²/ sec and 54.25%, respectively. Trypsin treatment increased D and %R to 6.4×10^–9 cm²/sec and 72.15%. In contrast, the anisotropy (r) for control cells was 0.270 which remained unchanged by trypsin treatment. The results show that diffusion of lipids in the plane of the membrane is restricted by trypsin-sensitive barriers.     

Effect of acute hypoxia on blood TSH levels.

The effect of simulated altitude produced by decompression chambers upon the thyroid function was studied in female rats. A significant decrease in blood TSH levels was found when the rats were maintained during 24 hours at a pressure of 0.4 atmosphere, but not at a pressure of 0.7 atmosphere.     

Effects of loaded breathing and hypoxia on diaphragm metabolism as measured by (31)P-NMR spectroscopy.

Diaphragm fatigue may contribute to respiratory failure. (31)P-nuclear magnetic resonance spectroscopy is a useful tool to assess energetic changes within the diaphragm during fatigue, as indicated by P(i) accumulation and phosphocreatine (PCr) depletion. We hypothesized that loaded breathing during hypoxia would lead to diaphragm fatigue and inadequate aerobic metabolism. Seven piglets were anesthetized by using halothane inhalation. Diaphragmatic contractility was assessed by transdiaphragmatic pressure (Pdi) at end expiration with the airway occluded. A nuclear magnetic resonance surface coil placed under the right hemidiaphragm measured P(i) and PCr during four conditions: control, inspiratory resistive breathing (IRB), IRB with hypoxia, and recovery (IRB without hypoxia). IRB alone resulted in hypercarbia (32 +/- 7 to 61 +/- 21 Torr) and respiratory acidosis but no change in diaphragm force output or aerobic metabolism. Combined IRB and hypoxia resulted in decreased force output (Pdi decreased by 40%; from 30 +/- 17 to 19 +/- 11 mmHg) and evidence of metabolic stress (ratio of P(i) to PCr increased by 290%; from 0.19 +/- 0.09 to 0.74 +/- 0.27). We conclude that diaphragm fatigue associated with inadequate aerobic oxidative metabolism occurs in the setting of loaded breathing and hypoxia. Conversely, aerobic metabolism and force output of the diaphragm remain unchanged from control during loaded normoxic or hyperoxic breathing despite the onset of respiratory failure.     

Effect of phalloidin on actin proteolysis as measured by viscometry and fluorimetry.

Even at low concentration, phalloidin shows a marked protection of F-actin against the action of trypsin or pronase. G-actin is not protected at any concentration of phallodin. The kinetics of the proteolysis show that a change in the environment of tryptophan residues is preceded by disruption of the filamentous structure of F-actin.     

Effects of fosmidomycin on plant photosynthesis as measured by gas exchange and chlorophyll fluorescence

In higher plants, many isoprenoids are synthesised via the chloroplastic 1-deoxy-d-xylulose 5-phosphate/2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Attempts to elucidate the function of individual isoprenoids have used the antibiotic/herbicidal compound fosmidomycin (3-[N-formyl-N-hydroxy amino] propyl phosphonic acid) to inhibit this pathway. Examination of the effect of fosmidomycin on the major components of photosynthesis in leaves of white poplar (Populus alba) and tobacco (Nicotiana tabacum) was made. Fosmidomycin reduced net photosynthesis in both species within 1 h of application, but only when photosynthesis was light-saturated. In P. alba, these reductions were confounded by high light and fosmidomycin inducing stomatal patchiness. In tobacco, this was caused by significant reductions in PSII chlorophyll fluorescence and reductions in V _cmax and J _max. Our data indicate that the diminution of photosynthesis is likely a complex effect resulting from the inhibition of multiple MEP pathway products, resulting in photoinhibition and photo-damage. These effects should be accounted for in experimental design and analysis when using fosmidomycin to avoid misinterpretation of results as measured by gas exchange and chlorophyll fluorescence.     

Cell membrane fluidity in the intact kidney proximal tubule measured by orientation-independent fluorescence anisotropy imaging.

Membrane fluidity was measured in the isolated perfused proximal tubule from rabbit kidney. The apical and basolateral plasma membranes of tubule cells were stained separately with the fluidity-sensitive fluorophore trimethylammonium-diphenyl-hexatriene (TMA-DPH) by luminal or bath perfusion. Fluorescence anisotropy (r) of TMA-DPH was mapped with spatial resolution using an epifluorescence microscope (excitation 380 nm, emission greater than 410 nm) equipped with rotatable polarizers and a quantitative imaging system. To measure r without the confounding effects of fluorophore orientation, images were recorded with emission polarizer parallel and perpendicular to a continuum of orientations of the excitation polarizer. The theoretical basis of this approach was developed and its limitations were evaluated by mathematical modeling. The tubule inner surface (brush border) was brightly stained when the lumen was perfused with 1 microM TMA-DPH for 5 min; apical membrane r was 0.281 +/- 0.006 (23 degrees C). Staining of the tubule basolateral membrane by addition of TMA-DPH to the bath gave a significantly lower r of 0.242 +/- 0.010 (P less than 0.005); there was no staining of the brush border membrane. To interpret anisotropy images quantitatively, effects of tubule geometry, TMA-DPH lifetime, fluorescence anisotropy decay, and objective-depolarization were evaluated. Steady-state and time-resolved r and lifetimes in the intact tubule, measured by a nanosecond pulsed microscopy method, were compared with results in isolated apical and basolateral membrane vesicles from rabbit proximal tubule measured by cuvette fluorometry; r was 0.281 (apical membrane) and 0.276 (basolateral membrane) (23 degrees C). These results establish a methodology to quantitate membrane fluidity in the intact proximal tubule, and demonstrate a significantly higher fluidity in the basolateral membrane than in the apical membrane.     

Comparison of X-ray fluorescence and optical fluorescence measured behind scattering layers as a basis for X-ray fluorescence endoscopy.

Recent developments in the technology of capillary-fiber optics suitable for X-rays in the range of approximately 4-10keV point to the possible realization of endoscopes applicable in X-ray fluorescence analysis. A general problem is the determination of scattering and absorption processes with consideration to tissue optics, X-ray scattering and X-ray absorption in a diagnostic partial volume. Therefore comparative investigations were performed in order to answer these questions. Zinc-oxide nanoparticles configured as single particles and ZnO clusters provided the fluorescence source in cell layers. An artificial scattering material was employed, which closely approximated the tissue optical conditions and the X-ray optical application conditions in possible diagnostic situations. As a result imaging of spatially resolved X-ray contrasts was better than adequate optical fluorescence imaging by approximately one magnitude. Hence a very important precondition for realizing X-ray fluorescence endoscopy is fulfilled.     

Nuclear envelope permeability measured by fluorescence microphotolysis of single liver cell nuclei

Fluorescence microphotolysis ("photobleaching") has been widely used to measure translational diffusion coefficients of lipids and proteins in cell membranes. This communication shows that fluorescence microphotolysis can be also employed for measurement of membrane transport in single cells and organelles. The influx of fluorescently labeled dextrans of graded molecular size into leaky human erythrocyte ghosts and isolated rat liver cell nuclei has been measured. For the nuclear envelope, a functional pore radius of 56-59 A is derived.     

Electrophoretic charge density and persistence length of DNA as measured by fluorescence microscopy

Individual ethidium-stained DNA molecules, embedded in an agarose gel made with electrophoresis buffer (0.05 molar salt), are observed using a fluorescence microscope. In the first experiment, open circular 66 kilobase pair (kbp) plasmids, immobilized by agarose fibers threaded through their centers, display entropic "rubber" elasticity. The charged molecules extend in an electric field of several volts per centimeter and contract to a compact random coil when the field is removed. The extension of the plasmids as a function of field strength is consistent with the freely jointed chain model when the effective electrophoretic charge density is set at 15 e-per persistence length. In a second experiment, stained linear 48.5 kbp DNA molecules are observed as random coils immobilized in agarose. A measure of their size, here named the "maximal-X-extent," is taken for 100 molecules and found to average 1.47 mu. A Monte Carlo computer simulation of random coils (freely jointed chain model) gives the same maximal-X-extent value when the persistence length is set at 0.08 mu.     

Proximity of lectin receptors on the cell surface measured by fluorescence energy transfer in a flow system.

Molecules of the lectin concanavalin A have been labeled separately with the fluorescein and rhodamine chromophores and jointly bound to the surface of transformed Friend erythroleukemia cells. The two dyes constitute an ideal donor-acceptor pair for fluorescence resonance energy transfer thereby permitting the determination of the proximity relationships between bound ligand molecules and the corresponding surface receptors. The transfer efficiency at saturation (about 57%) was measured in a multiparameter flow system using laser excitation at 488 nm and detection of fluorescein and rhodamine emission intensities as well as the emission anisotropy of the rhodamine fluorescence for each cell. The degree of energy transfer was estimated from the quenching of donor emission, the sensitization of acceptor emission, and the depolarization of acceptor fluorescence. The system has been modeled according to a formalism developed by Gennis and Cantor (Biochemistry 11: 2509, 1972). We estimate the separation between the surfaces of bound lectin molecules at saturation to be 0-40 A, a range possibly characteristic for micropatches induced by ligand binding.     

Conformational changes of the sweet protein monellin as measured by fluorescence emission

Monellin is a protein that tastes sweet. In the native state it is a dimer composed of two dissimilar noncovalently associated polypeptides. The conformation of the protein is a determinant of its sweetness, and the present investigation takes advantage of the fluorescence spectrum being a sensitive index of its conformation. The emission spectrum is used to evaluate the ability of temperature and pH to alter the conformation and the sweetness of the protein. When monellin dissolved in water is heated in discrete steps from 25 to 100 degrees C, its sweetness decreases. The halfwidth of the fluorescence emission band increases in parallel with the loss of sweetness. The increase in halfwidth is due primarily to an increase in the intensity of tyrosine emission that may be the result of the two dissimilar polypeptides of monellin beginning to separate. Tyrosine residues are present in both chains, while the single tryptophan occurs in only one. Monellin is less susceptible to denaturation by increasing temperature when dissolved in sodium acetate buffer at pH 4 than it is at pH 3 or 7. When the pH of a solution containing monellin in 0.1 M KC1 is varied from 2 to 13, there is a broad pH range (pH 4 to 9) where monellin's conformation is not markedly altered. Below pH 3.5 and above pH 10.5, however, the emission spectra indicate that substantial denaturation occurs. However, monellin can be partially renatured following pH 12 treatment with only minimal loss of sweetness. The sweetness of monellin under these two types of denaturing conditions, temperature and pH, can be predicted by the fluorescence emission spectrum of the protein. In addition, this study confirms that the biological activity of monellin, its sweetness, is a function of quaternary structure of the protein.     

Oligomeric states of the HIV-1 integrase as measured by time-resolved fluorescence anisotropy

Self-assembly properties of HIV-1 integrase were investigated by time-resolved fluorescence anisotropy using tryptophanyl residues as a probe. From simulation analyses, we show that suitable photon counting leads to an accurate determination of long rotational correlation times in the range of 20-80 ns, permitting the distinction of the monomer, dimer, and tetramer from higher oligomeric forms of integrase. The accuracy of correlation times higher than 100 ns is too low to distinguish the octamer from other larger species. The oligomeric states of the widely used detergent-solubilized integrase were then studied in solution under varying parameters known to influence the activity. In the micromolar range, integrase exists as high-order multimers such as an octamer and/or aggregates and a well-defined tetramer, at 25 and 35 degrees C, respectively. However, integrase is monomeric at catalytically active concentrations (in the sub-micromolar range). Detergents (NP-40 and CHAPS) and divalent cation cofactors (Mg(2+) and Mn(2+)) have a clear dissociative effect on the high multimeric forms of integrase. In addition, we observed that Mg(2+) and Mn(2+) have different effects on both the oligomeric state and the conformation of the monomer. This could explain in part why these two metal cations are not equivalent in terms of catalytic activity in vitro. In contrast, addition of Zn(2+) stimulates dimerization. Interestingly, this role of Zn(2+) in the multimerization process was evident only in the presence of Mg(2+) which by itself does not induce oligomerization. Finally, it is highly suggested that the presence of detergent during the purification procedure plays a negative role in the proper self-assembly of integrase. Accordingly, the accompanying paper [Leh, H., et al. (2000) Biochemistry 39, 9285-9294] shows that a detergent-free integrase preparation has self-assembly and catalytic properties different from those of the detergent-solubilized enzyme.