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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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).

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.

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.

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.

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.