NIR spectroscopic imaging to map hemoglobin + myoglobin oxygenation, their concentration and optical pathlength across a beating pig heart during surgery.

The purpose of this paper is to demonstrate that near-infrared (NIR) spectroscopic imaging can provide spatial distribution (maps) of the absolute concentration of hemoglobin + myoglobin, oxygen saturation parameter and optical pathlength, reporting on the biochemico-physiological status of a beating heart in vivo. The method is based on processing the NIR spectroscopic images employing a first-derivative approach. Blood-pressure-controlled gating compensated the effect of heart motion on the imaging. All the maps are available simultaneously and noninvasively at a spatial resolution in the submillimeter range and can be obtained in a couple of minutes. The equipment has no mechanical contact with the tissue, thereby leaving the heart unaffected during the measurement.

Rhodamine 800 as a near-infrared fluorescent deposition flow tracer in rodent hearts.

We investigated the use of a near-infrared (NIR) fluorescent dye, Rhodamine 800 (Rhod800, λ(exc) = 693 nm, λ(em) > 720 nm) as a flow-dependent molecular tracer for NIR spectroscopy and high-resolution cardiac imaging. Rhod800 accumulates in isolated mitochondria in proportion to the mitochondrial membrane potential (ΔΨ). However, in the intact myocardium, Rhod800 binding is ΔΨ-independent. Rat hearts were perfused in a Langendorff mode with Krebs-Henseleit buffer containing 45-nM Rhod800 at normal (100%), increased (150%), or reduced (50%) baseline coronary flow (CF) per gram, for 30 to 60 min. In a different group of hearts, the left anterior descending artery (LAD) was occluded prior to Rhod800 infusion to create a flow deficit area. Rhod800 deposition was analyzed by: 1. absorbance spectroscopy kinetics in the Rhod800-perfused hearts, 2. Rhod800 absorbance and fluorescence imaging in the short-axis heart slices, and 3. dynamic epicardial/subepicardial fluorescence imaging of Rhod800 in KCl-arrested hearts, with a spatial resolution of ∼ 200 µm. Rhod800 deposition was proportional to the perfusate volume (CF and perfusion time) and there was no Rhod800 loss during the washout period. In the LAD-ligated hearts, Rhod800 fluorescence was missing from the no-flow, LAD-dependent endocardial and epicardial/subepicardial area. We concluded that Rhod800 can be used as a deposition flow tracer for dynamic cardiac imaging.

Defects in myoglobin oxygenation in K(ATP)-deficient mouse hearts under normal and stress conditions characterized by near infrared spectroscopy and imaging.

BACKGROUND: Disruption of ATP-sensitive potassium (K(ATP)) channel activity results in the development of dilated cardiomyopathy in response to different forms of stress, likely due to the underlying metabolic defects. To further understand the role of Kir6.2-containing channels in the development of cardiac disease, we analysed the left ventricular (LV) wall oxygenation and the physiologic responses induced by acute stress in non-dilated Kir6.2(-/-) hearts. METHODS: Control (C57BL6) and Kir6.2(-/-) mouse hearts were perfused in constant flow Langendorff mode with Krebs-Henseleit buffer. Myocardial oxygenation was evaluated using a newly developed technique, near infrared spectroscopic imaging (NIRSI) of the myoglobin (Mb) oxygen saturation parameter (OSP, ratio of oxy- to total Mb). RESULTS: 2,4-dinitrophenol (DNP, 50-µM) and isoproterenol (0.1-µM) failed to produce a transient vasodilatory response and caused a significant diastolic pressure increase in Kir6.2(-/-) hearts. DNP strongly suppressed contractile function in both groups and induced severe mean OSP decreases in Kir6.2(-/-) hearts. Isoproterenol-induced decreases in OSP were similar despite the lack of contractile function stimulation in the Kir6.2(-/-) group. The index of OSP spatial heterogeneity (relative dispersion, RD) was lower by 15% in the Kir6.2(-/-) group at the baseline conditions. Recovery after stress caused reduction of RD values by 20% (DNP) and 8% (isoproterenol) in controls; however, these values did not change in the Kir6.2(-/-) group. CONCLUSIONS: 1) NIRSI can be used to analyse 2-D dynamics of LV oxygenation in rodent models of cardiomyopathy; 2) Kir6.2-containing K(ATP) channels play an important role in maintaining myocardial oxygenation balance under acute stress conditions and in post-stress recovery.

Fluorescence imaging to quantify the fluorescent microspheres in cardiac tissue.

To quantify the fluorescent microsphere (FM) content in cardiac tissue, which is an indicative of blood flow, fluorescence imaging of both sides of the pig heart slice was employed. Despite the light scattering inside the tissue and contributions from multiple tissue layers to the total emission, it is shown that the fluorescence intensity at any pixel is proportional to the FM content and the fluorescence image may be transformed to the image of the FM concentration. A convenient standard for the emission-FM concentration transformation is proposed. The approach has several advantages in comparison with the traditional "digestion & extraction" method such as: non-destructiveness, high spatial resolution, high throughput, repeatability and simplicity of operation.

Mapping the myoglobin concentration, oxygenation, and optical pathlength in heart ex vivo using near-infrared imaging.

A method that provides maps of absolute concentrations of oxygenated and deoxygenated myoglobin (Mb), its oxygenation, and its near-infrared (NIR) optical pathlength in cardiac tissue was developed. These parameters are available simultaneously. The method is based on NIR diffuse reflectance spectroscopic imaging and specific processing of the NIR images, which included a first derivative of the diffuse reflectance spectrum. Mb oxygenation, total Mb concentration, and NIR light pathlength were found to be in the range of 92%, 0.3 mM, and 12.5 mm, respectively, in beating isolated buffer-perfused and arrested pig hearts. The charge-coupled device camera enables sub-millimeter spatial resolution and spectroscopic imaging in 1.5 to 2.0 min. The technique is noninvasive and nondestructive. The equipment has no mechanical contact with the tissue of interest, leaving it undisturbed.

Characterization of cryoinjury-induced infarction with manganese-and gadolinium-enhanced MRI and optical spectroscopy in pig hearts.

PURPOSE: To investigate progression of cryoinjury in pigs using contrast-enhanced magnetic resonance imaging (MRI) as well as optical spectroscopy and imaging. METHODS: Cryoinjury was produced in 16 pigs in vivo and investigated using Gd-and Mn-enhanced MRI, optical imaging/spectroscopy and histology in acute and chronic setting up to 4 weeks after the injury. RESULTS: (1) Acute cryoinjury resulted in formation of a lesion with a severely reduced rate of sub-epicardial indocyanine green (intravascular optical flow tracer) passage. In vivo late Gd-enhanced MRI showed a approximately 10 mm deep hypointense area that was surrounded by a hyperintense rim while ex vivo Mn-enhanced MRI (MEMRI) detected a homogenous hypointense zone. Histological and spectroscopic examination revealed embolic erythrocytes blockages within the cryolesion with a thin necrotic rim neighboring the normal myocardium. (2) Chronic 4-week cryoinjury was characterized by uniform Gd-enhancement, whereas MEMRI revealed reduced Mn(2+)enhancement. Histological examination showed replacement of the cryoinjured myocardium by scar tissue. CONCLUSIONS: Acute cryoinjury resulted in formation of a no-reflow core embolized by erythrocytes and surrounded by a rim of necrotic tissue. Upon injury progression, the no-reflow zone shrunk and was completely replaced with scar tissue by 4 weeks after injury.

On the emission intensity of fluorescent microspheres in cardiac tissue images.

Formulations for the total fluorescence intensity of fluorescent microspheres in slabs of cardiac tissue were determined experimentally and theoretically. The tissue depth, at which the slab can be considered as a semi-infinite turbid medium, and critical layer thickness, which accounts for the most emission intensity were evaluated to be 8-9 and 3-5 mm, respectively, for the cardiac tissue. When fluorescent microspheres are linearly distributed across the slab depth, the mean absorption of them is proportional to the sum of their normalized total emissions in the slab excited from both sides. The formulations may be used for the fluorescence images analysis of cardiac and other biological tissues.

Hemoglobin plus myoglobin concentrations and near infrared light pathlength in phantom and pig hearts determined by diffuse reflectance spectroscopy.

To noninvasively determine absolute concentrations of hemoglobin (Hb) plus myoglobin (Mb) in cardiac tissue by means of regular near infrared (NIR) light diffuse reflectance measurements, a first derivative approach was applied. The method was developed to separately calculate oxygenated and deoxygenated [Hb+Mb] as well as an effective pathlength, which NIR light passes through in the tissue between optodes. Applying a cotton wool-based phantom, which mimics muscle tissue, it was shown that the intensity of the pseudo-optical density first derivative depends linearly on both oxygenated and deoxygenated Hb concentration, thereby validating the Lambert-Beer law in the range of 0 to 0.25 mM tetrameric Hb. A high correlation (R=0.995) was found between concentrations of Hb loaded onto the phantom and those determined spectrophotometrically, thereby verifying the first derivative method validity. The efficiency of the method was tested using in vivo pig hearts prior to and after ischemia initiated experimentally by left anterior descending artery branches occlusion. The results showed that the total [Hb+Mb] was 0.9-1.2 mM heme, the average tissue oxygen saturation was approximately 70% (which reduced to nearly 0% after occlusion), and the NIR (700-965 nm) light pathlength was 2.3 mm (differential pathlength factor [DPF]=2.7-2.8) in a living heart tissue.

Assessment of near-infrared path length in fibrous phantom and muscle tissue.

The first derivative of the pseudo-absorption spectrum of a water-loaded cotton wool (water-CW) phantom, which mimics muscle tissues, was used to determine the light path length in the near-infrared (NIR) region. The light path length increased as the density of the turbid medium decreased. It is independent of both water content in the range of 75-85% (by weight) and the diffuse reflecting reference used to determine the pseudo-absorbance. The path length determination procedure was verified by measurements of diffuse reflectance in chicken breast tissue for which the path length of 1.8 mm (differential path length factor, DPF = 2.1) was found to be similar to the path length of NIR light of 1.5-2.2 mm (DPF = 1.8-2.6) in a water-CW phantom of density similar to chicken breast. We conclude that the NIR light path length can serve as a characteristic of muscle tissue density.

Differential expression of secretory phospholipases A2 in normal and malignant prostate cell lines: regulation by cytokines, cell signaling pathways, and epigenetic mechanisms.

Upregulation of group IIA phospholipase A(2) (sPLA(2)-IIA) correlates with prostate tumor progression suggesting prooncogenic properties of this protein. Here, we report data on expression of three different sPLA(2) isozymes (groups IIA, V, and X) in normal (PrEC) and malignant (DU-145, PC-3, and LNCaP) human prostate cell lines. All studied cell lines constitutively expressed sPLA(2)-X, whereas sPLA(2)-V transcripts were identified only in malignant cells. In contrast, no expression of sPLA(2)-IIA was found in PrEC and DU-145 cells, but it was constitutively expressed by IFN-gamma in LNCaP and PC-3 cells. Expression of sPLA(2)-IIA is upregulated in PC-3 and in PrEC cell in a signal transducer and activator of transcription-1-dependent manner, but not in LNCaP cell. Additional signaling pathways regulating sPLA(2)-IIA expression include cAMP/protein kinase A, p38 mitogen-activated protein kinase, protein kinase C, Rho-kinase, and mitogen-activated/extracellular response protein kinase / extracellular signal-regulated kinase. No deletions were revealed in the sPLA(2)-IIA gene from DU-145 cells lacking the expression of sPLA(2)-IIA. Reexpression of sPLA(2)-IIA was induced by 5-aza-2'-deoxycytidine demonstrating that DNA methylation is implicated in the regulation of sPLA(2)-II. Together, these data suggest that sPLA(2)-IIA and sPLA(2)-V, but not sPLA(2)-X, are differentially expressed in normal and malignant prostate cells under the control of proinflammatory cytokines; epigenetic mechanisms appear involved in the regulation of sPLA(2)-IIA expression, at least in DU-145 cells.

Color of illumination during growth affects LHCII chiral macroaggregates in pea plant leaves.

To determine whether the color of illumination under which plants are grown, affects the structure of photosynthetic antennae, pea plants were grown under either blue-enriched, red-enriched, or white light. Carotenoid content of isolated chloroplasts was found to be insensitive to the color of illumination during growth, while chlorophyll a/b ratio in chloroplasts isolated from young illuminated leaves showed susceptibility to color. Color of illumination affects the LHCII chiral macroaggregates in intact leaves and isolated chloroplasts, providing light-induced alteration of the handedness of the LHCII chiral macroaggregate, as measured with circular dichroism and circularly polarized luminescence. The susceptibility of handedness to current illumination (red light excitation of chlorophyll fluorescence) is dependent on the color under which the plants were grown, and was maximal for the red-enriched illumination. We propose the existence of a long-term (growth period) color memory, which influences the susceptibility of the handedness of LHCII chiral macroaggregates to current light.

Left- and right-handed LHC II macroaggregates revealed by circularly polarized chlorophyll luminescence.

Circularly polarized chlorophyll luminescence (CPL) may serve as a measure of chiral macroaggregates of the light-harvesting chlorophyll-protein complexes (LHC II) in both isolated chloroplasts and intact leaves (Gussakovsky et al (2000) Photosynth Res 65: 83-92). In the present work, we applied the CPL approach to study the effect of fast (1-2 min) thermal impacts on LHC II macroaggregates. The results revealed unexpected temperature-response kinetics, composed of initial bell-shaped changes in the CPL signal, followed by degradation down to a steady state (equilibrium). The bell-shape effect was dependent upon illumination, and vanished in the dark. A mathematical analysis of the temperature-response kinetics uniquely indicated that LHC II chiral macroaggregates may persist in both left- and right-handed forms. These forms differ in their response to high temperatures. Both forms are more thermostable in leaves than in isolated chloroplasts. The cooperative degradation of LHC II macroaggregates, which is induced by the thermal impact, is irreversible. It is therefore suggested that the native LHC II macroaggregates are stable, stationary, non-equilibrium, spatially heterogeneous (dissipative) structures. The dissipative properties probably allow the interconversion between left- and right-handed forms under perturbation by certain factors. Illumination probably serves as one such perturbation factor, initiating the interconversion of dark-adapted, left-handed to light-dependent, right-handed LHC II macroaggregates. The chiral heterogeneity of the LHC II macroaggregates is a newly revealed aspect which needs to be taken into consideration in future circular dichroism or CPL studies.

Large-scale preparation of biologically active mouse and rat leptins and their L39A/D40A/F41A muteins which act as potent antagonists.

Expression plasmids encoding mouse and rat leptins and their L39A/D40A/F41A muteins were prepared. The proteins were expressed in Escherichia coli, refolded and purified to homogeneity, yielding electrophoretically pure, over 98% monomeric protein. Circular dichroism (CD) analysis revealed that the mutations hardly affect the leptins' secondary structure, and they were similar to previously reported CD spectra for human leptin. Both mouse and rat leptins were biologically active in promoting proliferation in BAF/3 cells stably transfected with the long form of human leptin receptor. The mutations did not change the binding properties to BAF/3 cells as compared, respectively, to non-mutated mouse, rat or human leptins, or their ability to form 1:1 complexes with the leptin-binding domain of chicken leptin receptor. In contrast, their biological activity, tested in a BAF/3 proliferation assay, was abolished and both became potent antagonists. As the LDF (amino acids 39-41) sequence is preserved in all known leptins, the present results substantiate the hypothesis that this sequence plays a pivotal role in leptins' site III and that interaction of leptin with its receptors resembles the corresponding interactions of interleukin-6 and granulocyte colony-stimulating factor their receptors.

Identification of the hydrophobic strand in the A-B loop of leptin as major binding site III: implications for large-scale preparation of potent recombinant human and ovine leptin antagonists.

Interaction of leptin with its receptors resembles that of interleukin-6 and granulocyte colony-stimulating factor, which interact with their receptors through binding sites I-III. Site III plays a pivotal role in receptors' dimerization or tetramerization and subsequent activation. Leptin's site III also mediates the formation of an active multimeric complex through its interaction with the IGD (immunoglobulin-like domain) of LEPRs (leptin receptors). Using a sensitive hydrophobic cluster analysis of leptin's and LEPR's sequences, we identified hydrophobic stretches in leptin's A-B loop (amino acids 39-42) and in the N-terminal end of LEPR's IGD (amino acids 325-328) that are predicted to participate in site III and to interact with each other in a beta-sheet-like configuration. To verify this hypothesis, we prepared and purified to homogeneity (as verified by SDS/PAGE, gel filtration and reverse-phase chromatography) several alanine muteins of amino acids 39-42 in human and ovine leptins. CD analyses revealed that those mutations hardly affect the secondary structure. All muteins acted as true antagonists, i.e. they bound LEPR with an affinity similar to the wild-type hormone, had no agonistic activity and specifically inhibited leptin action in several leptin-responsive in vitro bioassays. Alanine mutagenesis of LEPR's IGD (amino acids 325-328) drastically reduced its biological but not binding activity, indicating the importance of this region for interaction with leptin's site III. FRET (fluorescence resonance energy transfer) microscopy experiments have documented that the transient FRET signalling occurring upon exposure to leptin results not from binding of the ligand, but from ligand-induced oligomerization of LEPRs mediated by leptin's site III.

The effect of pea chloroplast alignment and variation of excitation wavelength on the circularly polarized chlorophyll luminescence.

Circularly polarized luminescence (CPL) is a powerful technique to study the macroorganization of photosynthetic light-harvesting apparatus in vivo and in vitro. It is particularly useful for monitoring environmental stress induced molecular re-organization of thylakoid membranes in green leaves. The current study focuses on two questions which are important to perform and interpret such experiments: how does CPL depend on the excitation wavelength and how on the orientation of the granal thylakoids. CPL and circular dichroism (CD) of pea chloroplasts were complementarily applied when chloroplasts were either in suspension or trapped in a polyacrylamide gel (PAAG) after alignment in a magnetic field. In contrast to the CD spectrum, the CPL signal was found to be independent of the excitation wavelength in both the Soret and the Qy absorption region for chloroplasts in both suspension and PAAG. The improved resolution of luminescence measurements revealed a relatively small negative CPL band in addition to the previously described large positive band. No effect of photoselection upon excitation on the CPL spectra was detected. The CPL intensity at 690 nm at the edge of the granal thylakoids was found to be higher than at the face of the grana suggesting the CPL anisotropy.

Watching proteins fold one molecule at a time.

Recent theoretical work suggests that protein folding involves an ensemble of pathways on a rugged energy landscape. We provide direct evidence for heterogeneous folding pathways from single-molecule studies, facilitated by a recently developed immobilization technique. Individual fluorophore-labeled molecules of the protein adenylate kinase were trapped within surface-tethered lipid vesicles, thereby allowing spatial restriction without inducing any spurious interactions with the environment, which often occur when using direct surface-linking techniques. The conformational fluctuations of these protein molecules, prepared at the thermodynamic midtransition point, were studied by using fluorescence resonance energy transfer between two specifically attached labels. Folding and unfolding transitions appeared in experimental time traces as correlated steps in donor and acceptor fluorescence intensity. The size of the steps, in fluorescence resonance energy transfer efficiency units, shows a very broad distribution. This distribution peaks at a relatively low value, indicating a preference for small-step motion on the energy landscape. The time scale of the transitions is also distributed, and although many transitions are too fast to be time-resolved here, the slowest ones may take >1 sec to complete. These extremely slow changes during the folding of single molecules highlight the possible importance of correlated, non-Markovian conformational dynamics.

Subcloning, expression, purification, and characterization of recombinant human leptin-binding domain.

A subdomain of the human leptin receptor encoding part of the extracellular domain (amino acids 428 to 635) was subcloned, expressed in a prokaryotic host, and purified to homogeneity, as evidenced by SDS-PAGE, with over 95% monomeric protein. The purified leptin-binding domain (LBD) exhibited the predicted beta structure, was capable of binding human, ovine, and chicken leptins, and formed a stable 1:1 complex with all mammalian leptins. The binding kinetics, assayed by surface plasmon resonance methodology, showed respective k(on) and k(off) values (mean +/- S.E.) of 1.20 +/- 0.23 x 10(-5) mol(-1) s(-1) and 1.85 +/- 0.30 x 10(-3) s(-1) and a K(d) value of 1.54 x 10(-8) m. Similar results were achieved with conventional binding experiments. LBD blocked leptin-induced, but not interleukin-3-induced, proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The modeled LBD structure and the known three-dimensional structure of human leptin were used to construct a model of 1:1 LBD.human leptin complex. Two main residues, Phe-500, located in loop L3, and Tyr-441, located in L1, are suggested to contribute to leptin binding.

Chiral macroaggregates of LHCII detected by circularly polarized luminescence in intact pea leaves are sensitive to drought stress.

Circularly polarized chlorophyll luminescence (CPL) was recently shown to be an effective tool for the study of chiral macroaggregate formation of the light-harvesting chlorophyll a/b pigment-protein complexes (LHCIIs) in isolated chloroplasts. The CPL measuring system was modified to study green leaves. Spectral and intensity features of pea leaf CPL signals suggested that CPL indeed detected chiral macroaggregates. The signals were found to depend on the excitation vs emission optical alignment, as well as on the side (adaxial or abaxial) of the leaf. Illumination of attached leaves with either low intensity or strong photoinhibitory light did not affect the chiral macroaggregation status. In contrast, the induction of drought stress in detached leaves led to full disruption of the chiral macroaggregates. The disruption developed gradually during slow dehydration, whereas under fast, heat-stimulated dehydration it was manifested in cooperative diminution of both CPL and photochemical capacity. Simultaneous exposure of the leaves to photoinhibitory illumination and fast dehydration resulted in negative CPL signals. The results demonstrate the potency of CPL as a non-destructive tool for structural studies of LHCII in intact leaves under varying environmental conditions.