Peak Fluorescence Intensity Research Articles

A Receptor-targeted Fluorescent Radiopharmaceutical for Multireporter Sentinel Lymph Node Imaging

To determine the imaging and receptor-binding properties of a multireporter probe designed for sentinel lymph node (SLN) mapping via nuclear and fluorescence detection. The animal experiments were approved by the institutional animal care and use committee. A multireporter probe was synthesized by covalently attaching cyanine 7 (Cy7), a near-infrared cyanine dye, to tilmanocept, a radiopharmaceutical that binds to a receptor specific to recticuloendothelial cells. In vitro binding assays of technetium 99m (99mTc)-labeled Cy7 tilmanocept were conducted at 4°C by using receptor-bearing macrophages. Optical SLN imaging after foot pad administration was performed by using two molar doses of Cy7 tilmanocept. Six mice were injected with 0.11 nmol of 99mTc-labeled Cy7 tilmanocept (low-dose group); an additional six mice were injected with 31 nmol of 99mTc-labeled Cy7 tilmanocept (high-dose group) to saturate the receptor sites within the SLN. After 2.5 hours of imaging, the mice were euthanized, and the sentinel and distal lymph nodes were excised and assayed for radioactivity for calculation of SLN percentage of injected dose and extraction. Four mice were used as controls for autofluorescence. Standard optical imaging software was used to plot integrated fluorescence intensity against time for calculation of the SLN uptake rate constant and scaled peak intensity. Significance was calculated by using the Student t test. In vitro binding assays showed subnanomolar affinity (mean dissociation constant, 0.25 nmol/L±0.10 [standard deviation]). Fluorescence imaging showed a detection sensitivity of 1.6×10(3) counts·sec(-1)·μW(-1) per picomole of Cy7. All four imaging metrics (percentage of injected dose, SLN extraction, SLN uptake rate constant, and expected peak fluorescence intensity) exhibited higher values (P=.005 to P=.042) in the low-dose group than in the high-dose group; this finding was consistent with receptor-mediated image formation. The multireporter probe 99mTc-labeled Cy7 tilmanocept exhibits in vitro and in vivo receptor-binding properties for successful receptor-targeted SLN mapping with nuclear and optical imaging.

A quantitative diagnostic method for oral mucous precancerosis by Rose Bengal fluorescence spectroscopy

A novel in vivo fluorescence spectroscopic diagnostic method has been developed in an animal model to make a quantified precancer diagnosis. In the study, 40 golden hamsters were randomly divided into four groups (groups A, B, C, and D), with group A being the control group and the other three groups being inducted at different precancer stages. A 1% Rose Bengal (RB) solution was used for the fluorescence spectroscopic diagnosis. A parameter K defined as K = I(RB)/I(auto) was introduced to reflect the amount of RB in the tissue, where I(RB) and I(auto) represent the fluorescence peak intensity of the RB in the tissue and the autofluorescence intensity of tissue at 580 nm, respectively. The average K values of the four groups were calculated and statistically analyzed by analysis of variance (ANOVA), which revealed statistically significant differences within each group as well as between groups (p < 0.001). After analysis by Clementine 11.1 C&R Tree modeling (CART), the following diagnostic criteria were set: normal, K ≤ 8.91; simple hyperplasia, 8.91 < K ≤ 41.92; mild dysplasia, 41.92 < K ≤ 70.79; moderate and severe dysplasia, K >70.79. The sensitivity and specificity to detect precancerous lesions compared with scalpel biopsy were calculated. The results of this study showed that the spectrofluorometric method mediated by RB could accurately discriminate different precancer stages.

Detection of Antibodies Against Major Histocompatibility Complex Class I-Related Chain A in Long-term Renal Graft Recipients

To determine the prevalence, allele specificity, and intensity of anti-MICA antibodies in long-term renal graft recipients and to investigate their association with impaired renal function. Sixty-eight long-term (> 10 y) renal graft recipients were divided into 2 groups: (1) patients with impaired renal function (serum creatinine ≥ 2 mg/dL, n=6); (2) patients with normal renal function (serum creatinine < 176.8 μmol/L, n=62). Anti-MICA antibodies were tested using Luminex single antigen beads assays and the frequency, specificity, and intensity of these antibodies were compared between 2 patient groups. MICA antibodies were detected in 33% of impaired renal function patients and 15% of normal renal function patients (P > .05). Anti-MICA*027 antibodies were found in 11.76% of patients, whereas antibody to MICA*012 was found in 2.94% of patients. Interestingly, among all antibody specificities, MICA*001,*004, *007, *009, *012, and *018 were found more frequently in impaired renal function patients than in normal renal function patients. The peak mean fluorescence intensity levels of MICA antibodies in impaired renal function patients were significantly higher than those in normal renal function patients (P < .05). Our data suggest that increased prevalence and intensity of anti-MICA antibodies are associated with impaired renal graft function in long-term renal graft recipients and some MICA antibodies might be more important than others in mediating graft rejection.

Layer-by-layer assembled smectite-polymer nanocomposite film for rapid fluorometric detection of aflatoxin B1

Aflatoxin B1 (AFB1) is a potent biological toxin produced by fungi Aspergillus flavus and Aspergillus parasiticus. Current quantification methods for AFB1 are mostly established on immunoaffinity columns which are both costly and labor intensive. Inspired by smectites’ high AFB1 adsorption capacity and affinity, we report the synthesis of a smectite-polyacrylamide (PAM) nanocomposite film for AFB1 detection and prove the feasibility of such a film for AFB1 quantification. A layer-by-layer assembly process was developed to achieve uniform morphology and thickness of the nanocomposite films on flat silicon substrates. Positive correlations between the peak fluorescence intensity of the adsorbed AFB1 and its concentration in the test solutions were obtained. The smectite-PAM nanocomposite film has shown similar AFB1 adsorption capabilities as the smectite, while exhibiting excellent structural stability in aqueous solutions. Our results suggest the feasibility of using the smectite-PAM nanocomposite film as a simple biosensor to achieve rapid fluorometric quantification of AFB1.

Study on effect of lipophilic curcumin on sub-domain IIA site of human serum albumin during unfolded and refolded states: A synchronous fluorescence spectroscopic study

Curcumin having pharmaceutical application as anti-oxidant, anti-inflammatory and anti-carcinogenic drug necessitates studying interaction of this molecule with native, unfolded and refolded state of human serum albumin (HSA), carrier protein in the blood. We proposed a simultaneous static and dynamic fluorescence quenching mechanism operating in the complex formation between HSA and curcumin. Location of curcumin in the close proximity of tryptophan with respect to tyrosine was further evident from the observation of two fold increase in rate of depletion of SFS intensity for tryptophan with respect to tyrosine in HSA in SFS spectrum. Alteration of SFS spectral peak position, electronic absorbance, fluorescence intensity and lifetime suggested chemical denaturation by urea expectedly unfold the protein molecule in the absence and presence of curcumin. Denatured HSA had similar fluorescence peak position and lifetime to that of l-tryptophan in the polar environment. During unfolding of HSA the spectral change of tyrosine and tryptophan was resolved through synchronous fluorescence spectra at two different Δλ in absence and presence of curcumin. It is found that curcumin remained bound to unfolded state of HSA and facilitated the process by pushing tryptophan moiety to more polar environment in the unfolded state. Dilution of the denatured protein by phosphate buffer reversibly refolded the sub-domain IIA, by also recovering fluorescence lifetime loss, but it was slow in the presence of curcumin. kq values indicate that curcumin–HSA complex is formed in the unfolded and refolded states as observed for native state.

Fluorescent quenching for biofilm extracellular polymeric substances (EPS) bound with Cu(II)

The Cu(II) binding properties of loosely bound extracellular polymeric substances (LBEPSs) and tightly bound EPSs (TBEPSs) extracted from biofilm samples at two apparent molecular weight (AMW) ranges, >14kDa and 1–14kDa, were investigated using three-dimensional excitation–emission matrix (EEM) fluorescence spectroscopy. The protein-like and aromatic protein fluorescence peaks were identified in EEM fluorescence spectra as peaks A and B, respectively. The intensities of peaks A and B were generally quenched when Cu(II) was bound with LBEPSs or TBEPSs with AMWs>14kDa at various pH levels. Conversely, for 1–14kDa EPSs, fluorescence intensities of peaks A and B were not quenched when Cu(II) was bound with LBEPSs at pH 4 or with both LBEPSs and TBEPSs at pH 8. The Stern–Volmer constant (logKsv) for the Cu(II)–LBEPSs and Cu(II)–TBEPSs binding processes were 2.38–4.37. The capability of EPSs to bind with Cu(II) increased as pH increased. At pH>4, the protein-like substances and aromatic proteins in TBEPSs had greater Cu(II) binding capability than LBEPSs. Additionally, the EPSs with AMWs>14kDa had stronger binding capability with Cu(II) than EPSs with AMWs of 1–14kDa. The difference in Cu binding behavior of LBEPS and TBEPS significantly affect the mobility, bioavailability, and toxicity of Cu in aquatic environments.

Complexion between mercury and humic substances from different landfill stabilization processes and its implication for the environment

Three-dimensional excitation emission matrix (EEM) fluorescence spectroscopy was employed to investigate the structural properties and Hg(II)-binding behavior of humic substances (HS) extracted from different landfill stabilization processes. The EEM fluorescence properties of humic acid (HA) are characterized by intense fluorescence at Ex/Em=440/500nm and Ex/Em=380/460nm. Two relatively strong fluorescence peaks appeared in the region of Ex/Em=260–290/350–370nm with the landfill time extended, which represented a protein-like or soluble microbial byproduct structure. The fluorescence EEM spectrum of fulvic acid (FA) featured a prominent peak of strong relative fluorescence intensity (FI=1598) at Ex/Em=330/440nm (peak C) accompanied by a weak fluorophore (FI=594) located at Ex/Em=275/445nm (peak D). There were strong interactions between HA and Hg, and the overall stability constant of Hg(II)–HA was mainly determined by the abundant O-ligands existing in HA. FA had a much higher Hg(II)-complexing capacity compared to HA samples, which may be ascribed to its relatively high content of carboxylic groups. The Hg(II)-complexing capacity of HA tended to decrease with stabilization process extension. The much higher Hg(II)-complexing capacity of FA than that of HA implied that FA played an important role in binding Hg(II) in early landfill stabilization process.

Fluorescence upconversion properties of a chiral polybinaphthyl induced by two‐photon absorption

AbstractThe fluorescence properties of a chiral polymer based on optically active polybinaphthyls were studied in tetrahydrofuran solution. One‐photon excited fluorescence of the polymer was located in the range of ∼ 596 nm and the corresponding lifetime was ∼ 4.38 ns. From the excitation spectra and emission spectra excited at 800 nm, the upconversion fluorescence emission was observed. When excited by using 800 nm fs laser pulses with different input irradiances, the peak fluorescence intensity of the solution provides square dependence with the input irradiance power, giving an evidence for two‐photon excited fluorescence. Furthermore, open aperture Z‐scan measurements were performed at different irradiation intensities to confirm two‐photon absorption property of the solution at 800 nm excitation. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

The period effects of intraperitoneal administration of different gold nanoparticle sizes on heart tissue of rats using fluorescence measurements: In vivo

Despite many benefits of nanotechnology, some studies indicate that certain nanoparticle (NPs) may cause adverse effects because of their small size and unique properties. The aim of the present study was to elucidate the period effects of intraperitonealy administration of different gold nanoparticle sizes on rat heart tissue in vivo using fluorescence spectroscopy. The experimental rats were divided into control and six groups (G1A, G1B, G2A, G2B, G3A and G3B; G1: 20 nm; G2: 10 nm; G3: 50 nm; A: infusion of GNPs for 3 days; B: infusion of GNPs for 7 days). To investigate the period effects of gold nanoparticles (GNPs) 10, 20 and 50 nm on the heart tissue of rats, 50 µl dose of GNPs (of concentration 0.1% Au) were intraperitonealy injected into rats for periods of 3 and 7 days to identify the toxicity and tissue distribution of GNPs in vivo using fluorescence measurements. The high electron densities of GNPs as well as the homogeneity of the particles shape and size make them highly conspicuous under the transmission electron microscope (TEM). The peak fluorescence intensity increased for G1B compared with G1A, increased for G2B compared with G2A and sharply decreased for G3A and G3B compared with the control. The peaks of G1A, G1B, G2A, G2B, G3A and G3B shifted towards the UV-Visible wavelength compared with the control. The peak fluorescence intensity for G1A was higher than G2A, G3A and G3B while G2A was higher than G3A. The peak fluorescence intensity for G2B was higher than G1B and G3B while G1B was higher than G3B. GNPs of sizes 10 and 20 nm have spherical shape while GNPs of size 50 nm have hexagonal shape. Fluorescence intensity of GNPs was size, shape and infusion period dependent. The decrease in peak fluorescence intensity induced in large 50 nm GNPs may be attributed to occurrence of quenching, decrease number and surface area of GNPs in addition to high clearance of GNPs via urine and bile. Moreover, decreasing size may lead to an exponential increase in surface area relative to volume, thus making the GNPs surface more reactive on itself (aggregation) and to its surrounding environment (biological components). Size, shape, surface area, number and clearance of GNPs play a key role in toxicity, and alterations of accumulation of GNPs in the heart tissue which may be mediated by dynamic protein binding and exchange. A better understanding of these mechanisms will improve drug delivery. Key words: Gold nanoparticles, sizes, administration period, heart tissue, fluorescence spectroscopy.

The period effects of intraperitoneal administration of different gold nanoparticle sizes on kidney tissue of rat in vivo using fluorescence measurements

Despite the many benefits of nanotechnology, some studies indicate that certain nanoparticles may cause adverse effects because of their small size and unique properties. The aim of the present study was to elucidate the period effects of intraperitoneal administration of different gold GNP sizes on the rat kidney tissue in vivo using fluorescence spectroscopy. The experimental rats were divided into control and six groups (G1A, G1B, G2A, G2B, G3A and G3B; G1: 20 nm; G2: 10 nm; G3: 50 nm; A: infusion of GNPs for 3 days; B: infusion of GNPs for 7 days). To investigate the period effects of GNPs 10, 20 and 50 nm on the kidney tissue of rats, 50 µl dose of GNPs (of concentration 0.1% Au) were intraperitonealy injected into rats for periods of 3 and 7 days to identify the toxicity and tissue distribution of GNPs in vivo using fluorescence measurements. GNPs of sizes 10 and 20 nm show spherical morphology with good particle size distribution dispersed in the solution while GNPs of size 50 nm have no spherical shape, but they have hexagonal shape. At the infusion period of 3 days, the fluorescence intensity of the 1st peaks increased for G1A and decreased for G2A and G3A compared with the control while the fluorescence intensity of the 2nd peaks decreased for G1A, G2A and G3A compared with the control. At the infusion period of 7 days, the fluorescence intensity of the 1st and 2nd peaks increased for G1B and G2B and sharply decreased for G3B compared with the control. At the infusion periods of 3 and 7 days, the fluorescence intensity of the 1st peaks increased for G1A, G1B and G2B and decreased for G2A, G3A and G3B compared with the control while the fluorescence intensity of the 2nd peaks decreased for G1A, G1B, G2A, G2B and G3A compared with the control. Fluorescence intensity of GNPs varied with the GNP size. The decrease in fluorescence intensity may be attributed to size, shape, number of GNPs, quenching of 50 nm GNPs, surface area of GNPs and slow clearance for GNPs of 10 and 20 nm through urine and bile from the kidney. Moreover, decreasing size may lead to an exponential increase in surface area relative to volume, thus making the GNPs surface more reactive on itself (aggregation) and to its surrounding environment (biological components). Size and shape of GNPs may be related to their useful characters and also plays a key role in toxicity. The alterations of accumulation in the kidney tissue, depending on GNP size, which may be mediated by dynamic protein binding and exchange. A better understanding of these mechanisms will improve drug delivery and period and dose estimation used in the risk assessment. Key words: Gold nanoparticles, sizes, period effects, kidney tissue, fluorescence spectroscopy.

Polarization-induced control of two-photon excited fluorescence in a chiral polybinaphthyl

The fluorescence behavior of a chiral polybinaphthyl excited with 100 fs 800 nm laser pulses was investigated in tetrahydrofuran solution. The peak fluorescence intensity versus the input irradiance was measured to meet a square dependence, giving evidence for two-photon excited fluorescence (TPF). The variations of the TPF intensity were found to be strongly modulated by the different polarized incident lights and tightly depend on the linearly polarized component of the incident light. Furthermore, combining with the characteristics of chiral molecules, the two-photon polarization ratio was studied to reveal the symmetry of the involved excited states.

The intrinsic fluorescence spectrum of dilute gastric juice as a novel diagnostic tool for gastric cancer

To investigate the intrinsic fluorescence spectrum of gastric juice as a diagnostic method for gastric cancer. We collected gastric juice by gastroscopy in 1,870 patients from May 2001 to March 2006, of whom 202 were involved in a preliminary test, 162 in experimental optimization and 1,506 in clinical verification. The best dilution and pH value were chosen in the experimental optimization phase. Clinical verification was based on optimized samples. Intrinsic fluorescence spectra were measured in all samples with a fluorescence spectrophotometer using an excitation wavelength of 288 nm. The first peak of fluorescence intensity (P(1) FI) of the intrinsic fluorescence spectrum was significantly higher in gastric juice from patients with gastric cancer than from those with benign lesions. There was no significant difference in the P(1) FI differences between patients with benign and malignant lesions with samples diluted by 20-fold to 80-fold and from pH 9 to pH 11. Clinical verification in 1,506 patients showed that P(1) FI ≥ 76.5 was the optimal cut-off on the receiver operating characteristic curve for diagnosing gastric cancers: sensitivity was 83.2%, specificity 80.7% and accuracy 82.0%. P(1) FI of the intrinsic fluorescence at 288 nm is significantly higher in patients with gastric cancers than in individuals with benign lesions. As a clinical indicator of gastric cancer, its sensitivity, specificity and accuracy were high.

Characterisation of dissolved organic matter in karst spring waters using intrinsic fluorescence: Relationship with infiltration processes

From analysis of spectrophotometric properties of dissolved organic matter (OM) and the hydrochemical responses of some karst springs under different hydrologic conditions, an assessment of the origin and transfer pathway of OM present in karst spring waters, from soil and epikarst toward the spring, has been conducted for three karst aquifers in southern Spain: Alta Cadena, Sierra de Enmedio and Los Tajos. Intrinsic fluorescence (excitation–emission matrices or EEMs), together with major water chemistry (electrical conductivity, temperature, alkalinity, Cl−, Mg+2) and PCO2 along with natural hydrochemical tracers (TOC and NO3−), have been monitored in 19 springs which drain the three karst aquifers examined in this study. The spring water EEM spectra indicate that fulvic acid-like substances, produced in the soil as a consequence of the decomposition of OM, are the dominant fluorophores, although some of the OM appears to originate from in situ microbiological activity but could be indicative of contamination present in recharge waters from livestock. During each recharge event, TOC and NO3− concentrations increased and variations in fluorescence intensities of peaks attributed to fulvic acid-like compounds were observed. In areas with minimal soil development, spatial and temporal variations in the fluorescence intensity of fulvic acid-like substances and other fluorophores derived from microbiological activity, together with other hydrochemical parameters, provide insights into the hydrogeological functioning of karst aquifers and the infiltration velocity of water from soil and facilitate assessment of contamination vulnerability in these aquifers.

Validation and quantification of extractable age pigments for determining the age of Antarctic krill (Euphausia superba)

Antarctic krill, Euphausia superba, hatched from eggs and maintained for four years, were sampled periodically for age-pigment analysis. Extractable pigments from the eye and eyestalk ganglia were quantified using fluorescence intensity and standardised against protein. Three peak fluorescence intensities were detected at wavelengths of excitation 280 nm, emission 625 nm (pigment 1); excitation 355 nm, emission 510 nm (pigment 2); and excitation 463 nm, emission 620 nm (pigment 3). There was a positive correlation between the quantity of pigments 1 and 3 and the age of Antarctic krill. A model was developed to predict age from pigment 3 and to compare it with other age proxies (carapace length and eyeball diameter). The quantity of pigment 3 was the best predictor of age. The pigment method can discriminate between similar sized krill aged 12 and 36 months. Age pigments provide an improved tool for age estimation in Antarctic krill, particularly if used in conjunction with other demographic information.

Novel insights into destruction mechanisms in a hybrid membrane process for simultaneous sludge thickening and digestion by characterization of dissolved organic matter

Three-dimensional excitation–emission matrix (EEM) fluorescence spectroscopy and gel filtration chromatography (GFC) were employed to characterize dissolved organic matter (DOM) in a hybrid membrane process for simultaneous sludge thickening and digestion (MSTD). The MSTD process was operated under a sequencing batch mode, and the operating time of each cycle was 15 d from the initial fresh sludge to the final thickened and digested sludge. Results showed that the molecular weight (MW) distribution of DOM in sludge supernatant became broader after the MSTD process, and three main peaks including Peak A and Peak B related to protein-like substances and Peak C associated with humic acid-like substances could be identified from the EEM fluorescence spectra of all DOM samples in the MSTD process. The quantitative analysis of the fluorescence spectra of DOM in sludge supernatant showed that the fluorescence intensity (FI) of Peak A and Peak B in sludge supernatant correlated well with the concentration of soluble protein, MLSS destruction efficiency and mean particle size, suggesting that the variations of EEM fluorescence spectra of DOM in sludge supernatant could be used to evaluate the floc destruction mechanisms of the MSTD process. It was also found that the FI of Peak A slightly increased before 8 d but rapidly went up after 8 d, and the FI of Peak B changed slightly before 8 d but increased after 8 d, which implied that the floc destruction mechanisms were varied in one cycle of the MSTD process.

A Novel Pyridoxal 5′-Phosphate-dependent Amino Acid Racemase in the Aplysia californica Central Nervous System

D-aspartate (D-Asp) is found in specific neurons, transported to neuronal terminals and released in a stimulation-dependent manner. Because D-Asp formation is not well understood, determining its function has proved challenging. Significant levels of D-Asp are present in the cerebral ganglion of the F- and C-clusters of the invertebrate Aplysia californica, and D-Asp appears to be involved in cell-cell communication in this system. Here, we describe a novel protein, DAR1, from A. californica that can convert aspartate and serine to their other chiral form in a pyridoxal 5'-phosphate (PLP)-dependent manner. DAR1 has a predicted length of 325 amino acids and is 55% identical to the bivalve aspartate racemase, EC 5.1.1.13, and 41% identical to the mammalian serine racemase, EC 5.1.1.18. However, it is only 14% identical to the recently reported mammalian aspartate racemase, DR, which is closely related to glutamate-oxaloacetate transaminase, EC 2.6.1.1. Using whole-mount immunohistochemistry staining of the A. californica central nervous system, we localized DAR1-like immunoreactivity to the medial region of the cerebral ganglion where the F- and C-clusters are situated. The biochemical and functional similarities between DAR1 and other animal serine and aspartate racemases make it valuable for examining PLP-dependent racemases, promising to increase our knowledge of enzyme regulation and ultimately, D-serine and D-Asp signaling pathways.

Optical properties of dyes with/without metal nanoparticles doped in a highly ordered nanostructure

Highly ordered nanocomposite arrays of Rh6G-Au-AAO are formed by filling anodized aluminum oxide (AAO) with Rhodamine 6G (Rh6G) and gold nanoparticles. The optical properties of Rh6G-Au-AAO are studied by visible absorptive and fluorescent spectroscopy. Compared with the fluorescence spectra of Rh6G-Au in the solution environment, the fluorescence peak intensities of Rh6G-Au-AAO are significantly enhanced, the maximum enhancement rate is 5.5, and a constant blue shift of ∼12 nm of peak positions is presented. The effects come from the spatial confinement of AAO and the inhibition of the fluorescence quenching effect induced by gold nanoparticles. The results show that the nanocomposite structures of fluorescence molecules-metal nanoparticles-AAO have a considerable potential in engineering molecular assemblies and creating functional materials of superior properties for future nanophotonics.

Activation of CFTR trafficking and gating by vasoactive intestinal peptide in human bronchial epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is an apical membrane chloride channel critical to the regulation of fluid, chloride, and bicarbonate transport in epithelia and other cell types. The most common cause of cystic fibrosis (CF) is the abnormal trafficking of CFTR mutants. Therefore, understanding the cellular machineries that transit CFTR from the endoplasmic reticulum to the cell surface is important. Vasoactive intestinal polypeptide (VIP) plays an important role in CFTR-dependent chloride transport. The present study was designed to observe the affection of VIP on the trafficking of CFTR, and channel gating in human bronchial epithelium cells (HBEC). Confocal microscopy revealed CFTR immunofluorescence extending from the apical membrane deeply into the cell cytoplasm. After VIP treatment, apical extension of CFTR immunofluorescence into the cell was reduced and the peak intensity of CFTR fluorescence shifted towards the apical membrane. Western blot showed VIP increased cell surface and total CFTR. Compared with the augmented level of total CFTR, the surface CFTR increased more markedly. Immunoprecipitation founded that the mature form of CFTR had a marked increase in HBEC treated with VIP. VIP led to a threefold increase in Cl(-) efflux in HBEC. Glibenclamide-sensitive and DIDS-insensitive CFTR Cl(-) currents were consistently observed after stimulation with VIP (10(-8) mol/L). The augmentation of CFTR Cl(-) currents enhanced by VIP (10(-8) mol/L) was reversed, at least in part, by the protein kinase A (PKA) inhibitor, H-89 and the protein kinase C (PKC) inhibitor, H-7, suggesting PKA and PKC participate in the VIP-promoted CFTR Cl(-) currents.

Mechanics and Function of Protein Clustering and Spatial Sorting at Cell Membranes

Cell signalling regulated by the spatial arrangement of proteins into clusters ranging in size from a few molecules up to micron-scale assemblies is now heavily studied. Although the timing, placement, and even the molecular details of the proteins involved may be well known, their exact mechanism of formation and function still elude researchers. We seek to answer these questions in the context of the immunological synapse (IS) using a live T cell-supported membrane system. The IS is a T cell-antigen presenting cell (APC) interaction wherein membrane receptor-ligand pairs are reorganized into concentric patterns. In its simplest form, T cell receptors (TCR) bound to peptide-presenting major histocompatibility complex (pMHC) form a central cluster surrounded by a ring of leukocyte function associated antigen-1 (LFA-1) bound to intercellular adhesion molecule-1 (ICAM-1). Both ligand-bound TCR and LFA-1 are known to be centripetally transported by the actin cytoskeleton, but how they are differentially sorted is unknown. We hypothesize that protein cluster size prior to transport regulates sorting and probe this by increasing the LFA-1 cluster size. To do this, we use a bivalent or tetravalent crosslinker against LFA-1 or its ICAM-1 ligand and compare the distribution to non-crosslinked counterparts in the same cell. Peak fluorescence intensities across the IS averaged for whole cell populations reveal increased centripetal LFA-1 transport with increased size until LFA-1 forms a central cluster like TCR. Based on our results, we propose a frictional force coupling model as the mechanism of IS formation. Moreover, using fluorescence correlation spectroscopy, we discover a gradient of LFA-1:ICAM-1 cluster sizes across the synapse, as predicted by our model.

Three-dimensional fluorescence spectral characterization of soil dissolved organic matters in the fluctuating water-level zone of Kai County, Three Gorges Reservoir

Three-dimensional fluorescence spectroscopy was used to investigate the fluorescent properties of soil dissolved organic matter (DOM) in the water-level-fluctuation zone (WLFZ) of Kai County, Three Gorges Reservoir (TGR). Most of the soil DOM analyzed in this study was found to contain four fluorescence peaks. Peaks A and C represent humic-like fluorescence, whereas peaks B and D represent tryptophan-like fluorescence. Peaks E and F, which represent tyrosine-like fluorescence, only appeared in certain soils. Soil humus was the main source of DOM in soil, and higher concentration of soil DOM was found in the exposed soil than submerged soil. Compared to the peaks A and B, the fluorescence intensities of peaks C and D were strongly influenced by the fluctuating water level. Analysis of fluorescence intensities of different peaks in soil DOM showed that WLFZ soil was not contaminated significantly. Soil DOM contained at least two types of humic-like fluorescence groups and two types of protein-like fluorescence groups. The proportion of the content of peak A in soil organic matter was quite stable. The soil DOM in exposed soil had relatively high humification and aromaticity, and periodic submerging and exposure of soil had an impact on the humification of soil DOM.