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Results for “cell deconvolution”

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At least 271 records · Page 15Linked to original sources

PubMed2004

Cisplatin rapidly down-regulates its own influx transporter hCTR1 in cultured human ovarian carcinoma cells.

PURPOSE: Cisplatin (DDP)-resistant cells commonly exhibit reduced drug accumulation. Previous studies have shown that the major copper (Cu) influx transporter CTR1 controls the uptake of DDP in yeast and mammalian cells. The goal of this study was to examine the effect of Cu and DDP on the level and subcellular localization of hCTR1 protein in human ovarian carcinoma cells. EXPERIMENTAL DESIGN: Cultured human ovarian carcinoma A2780 cells were exposed to DDP and Cu, and the effect on hCTR1 was determined using Western blot analysis and confocal digital deconvolution microscopy. RESULTS: Loss of hCTR1 was triggered by DDP exposure in a concentration and time-dependent manner. Exposure to 0.5 micromol/L DDP for 5 minutes reduced hCTR1 levels and exposure to DDP concentrations > or =2 micromol/L caused almost complete disappearance. The loss of hCTR1 was observed within 1 minute of the start of exposure to 2 micromol/L DDP. Treatment of cells with 100 micromol/L Cu for 5 minutes produced a smaller effect. Pretreatment of cells with 2 micromol/L DDP for 5 minutes resulted in a 50% decrease in 64Cu uptake, demonstrating that the DDP-induced loss of hCTR1 detected by Western blot analysis and imaging was functionally significant. CONCLUSIONS: DDP down-regulated the amount of its major influx transporter in cultured human ovarian carcinoma cells in a concentration- and time-dependent manner. The effect was observed at DDP concentrations within the range found in the plasma of patients being treated with DDP, and it occurred very quickly relative to the half-life of the drug.

Antineoplastic Agents↗

PubMed2002

Cell surface heparan sulfate is a receptor for attachment of envelope protein-free retrovirus-like particles and VSV-G pseudotyped MLV-derived retrovirus vectors to target cells.

Non-infectious, envelope protein-free, retrovirus-like particles (VLP) derived from either Moloney murine leukemia virus (MLV) or human HIV are able to bind efficiently to, but not infect, target cells. Upon subsequent addition to the bound particles of the G protein of vesicular stomatitis virus (VSV-G), an efficient surrogate retrovirus envelope protein, the VLP are efficiently taken up by the cells to produce infection. Cell attachment of the VLP is efficiently inhibited by soluble heparin and dextran sulfate and less efficiently abrogated by several other glycosaminoglycans (GAGs) including chondroitin sulfate A and chondroitin sulfate B (dermatan sulfate), as determined by deconvolution microscopic immunodetection of the viral gag protein and by quantitative binding studies of metabolically labeled (35)S-VLP. Enzymatic digestion of heparan sulfate (HS) from the cell surface with heparinase I also reduces VLP binding. Furthermore, VLP adsorption onto several CHO cell lines variably deficient in cell surface GAG is significantly but incompletely abrogated. De-sulfated heparins are less efficient than native heparin in inhibiting the Polybrene-mediated binding of VLP, whereas growth of human cells in the presence of sodium chlorate leads to significant reduction of Polybrene-mediated VLP binding. In addition, specific inhibition of VLP binding and infectivity of mature infectious VSV-G-pseudotyped virus is observed in the presence of heparin and HS under Polybrene-free conditions. We conclude from these studies that the presence of Polybrene, the degree of sulfation of cell surface GAG, and possibly the presence of charged cell surface macromolecules create an electrostatic environment that promotes optimum binding of VLP to cells. Additionally, our results demonstrate that, in the absence of Polybrene, initial attachments of non-infectious, envelope protein-free VLP and probably mature infectious virus particles are mediated by interactions of the virus particles with cell surface heparan sulfate, and possibly with other GAG molecules.

Animals↗

PubMedDate not supplied

Validation of deconvolutional analysis for the measurement of hepatic function in dogs with toxic-induced liver disease.

The extraction of the hepatobiliary radiopharmaceutical 99mTc-mebrofenin (Choletec) by the liver can be used to evaluate the severity of hepatocellular disease. The hepatic parenchymal cells extract mebrofenin from the blood by the same active transport mechanism as bilirubin. The ability of the liver to extract 99mTc-mebrofenin is a measure of hepatic parenchymal cell function. In this study, we induced hepatocellular disease by administration of a hepatotoxic drug and compared a direct method of determining the hepatic extraction of 99mTc-mebrofenin to hepatic extraction fraction derived from deconvolutional analysis. We also compared both methods of calculating the hepatic extraction of 99mTc-mebrofenin to liver histopathology. Hepatic extraction fraction derived from deconvolutional analysis correlated very well to the direct measurement technique (R=0.922, p < 0.001). Both methods of determining hepatic extraction correlated well to quantitative histopathology, having the same correlation coefficient and p values. (R=-0.833, p=0.003). As the hepatic extraction 99mTc-mebrofenin decreased, the severity of the histopathologic lesions of the liver increased in a linear fashion. There was a significant correlation of the hepatic excretion T1/2 to quantitative histopathology (R=0.949, p < 0.001). The hepatic excretion T1/2 increased as the severity of the histopathologic lesions of the liver increased. Hepatic extraction (HEF) and excretion of 99mTc-mebrofenin are good predictors of the severity of hepatocellular damage in toxic induced liver disease. This study helps validate the premise that HEF derived from deconvolutional analysis is a good predictor of the actual first pass hepatic extraction of 99mTc-mebrofenin.

Aniline Compounds↗

PubMed2004

Determination of uronic acids in isolated hemicelluloses from kenaf using diffuse reflectance infrared fourier transform spectroscopy (DRIFTS) and the curve-fitting deconvolution method.

Hemicellulose samples were isolated from kenaf (Hibiscus cannabinus L.). Hemicellulosic fractions usually contain a variable percentage of uronic acids. The uronic acid content (expressed in polygalacturonic acid) of the isolated hemicelluloses was determined by diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and the curve-fitting deconvolution method. A linear relationship between uronic acids content and the sum of the peak areas at 1745, 1715, and 1600 cm(-1) was established with a high correlation coefficient (0.98). The deconvolution analysis using the curve-fitting method allowed the elimination of spectral interferences from other cell wall components. The above method was compared with an established spectrophotometric method and was found equivalent for accuracy and repeatability (t-test, F-test). This method is applicable in analysis of natural or synthetic mixtures and/or crude substances. The proposed method is simple, rapid, and nondestructive for the samples.

Journal Article↗

PubMed2003

Characterization of GLP-1 effects on beta-cell function after meal ingestion in humans.

OBJECTIVE: Glucagon-like peptide 1 (GLP-1) is an incretin that augments insulin secretion after meal intake and is developed for treatment of type 2 diabetes. As a novel therapeutic agent, characteristics of its beta-cell effects are important to establish. Previously, beta-cell effects of GLP-1 have been characterized in humans during graded intravenous infusions of glucose, whereas its effects after more physiological stimuli, like meal intake, are not known. RESEARCH DESIGN AND METHODS: Eight women (aged 69 years, fasting glucose 3.7-10.3 mmol/l, BMI 22.4-43.9 kg/m(2)) who had fasted overnight were served a breakfast (450 kcal) with intravenous infusion of saline or synthetic GLP-1 (0.75 pmol x kg(-1) x min(-1)), and beta-cell function was evaluated by estimating the relationship between glucose concentration and insulin secretion (calculated by deconvolution of C-peptide data). RESULTS: -GLP-1 markedly augmented insulin secretion, despite lower glucose. Total insulin secretion was 29.7 +/- 4.2 nmol/m(2) with GLP-1 versus 21.0 +/- 1.6 nmol/m(2) with saline (P = 0.048). GLP-1 increased the dose-response relationship between glucose concentration and insulin secretion (70 +/- 26 with GLP-1 versus 38 +/- 16 pmol insulin. min(-1 x m(2). mmol(-1) glucose. l without, P = 0.037) and augmented the potentiation factor that modulates the dose response (2.71 +/- 0.42 with GLP-1 versus 0.97 +/- 0.17 without, P = 0.005). The potentiation factor correlated to GLP-1 concentration (r = 0.53, P < 0.001); a 10-fold increase in GLP-1 levels produced a twofold increase in the potentiation factor. These effects of GLP-1 did not correlate with fasting glucose levels or BMI. CONCLUSIONS: Administration of GLP-1 along with ingestion of a meal augments insulin secretion in humans by a dose-dependent potentiation of the dose-response relationship between plasma glucose and insulin secretion.

Aged↗

PubMedDate not supplied

Specific regulatory actions of dihydrotestosterone and estradiol on the dynamics of FSH secretion and clearance in humans.

The authors investigated immunoactive and bioactive follicle-stimulating hormone (FSH) secretion and clearance in six healthy young men during steady-state infusions of vehicle (basal, B, 28 hours), dihydrotestosterone (DHT, 4.5 days), or estradiol (4.5 days) accompanied by blood sampling at 10-minute intervals for 28 hours. Serum FSH concentrations were assayed by a two-site immunoradiometric assay (IRMA) and two separate in vitro bioassays (rat granulosa and Sertoli cell systems). FSH measurements included: 24-hour mean serum concentrations (IRMA and bioassay), multiple-parameter deconvolution of 24-hour pulsatile FSH time series and FSH release in response to exogenous gonadotropin-releasing hormone (GnRH) boluses (IRMA) to assess secretion and clearance, and circadian serum FSH concentration rhythms by cosinor analysis (IRMA). We found: 1) a significant decrease in 24-hour mean IRMA FSH concentrations during DHT infusion while both in vitro estimates of FSH bioactivity were unchanged; 2) significant decreases in the mass of IRMA FSH secreted per 24 hours during DHT infusion; 3) significant decreases in the IRMA FSH half-life during estradiol infusion without any change in FSH interpulse interval; 4) no steroidal effects on FSH secretory responses to exogenous GnRH; and 5) abolition of basal circadian FSH rhythms during sex-steroid infusions. Based on these findings, we conclude that steady-state sex-steroid hormone infusions selectively alter IRMA FSH secretion and clearance without affecting IRMA FSH pulse frequency or mean concentrations of bioactive FSH.

Adult↗

PubMed2005

S-cones do not contribute to the OFF-midget pathway in the retina of the marmoset, Callithrix jacchus.

It is well established that in primate retina both medium- and long-wavelength-sensitive cone types provide input to the midget-parvocellular pathway. The question, however, whether short-wavelength-sensitive (S or 'blue') cones provide input to the OFF-division of the midget-parvocellular pathway is still controversial. In the present study, we investigated the connections of nearly 400 S-cones with OFF-midget bipolar cells in central and peripheral retina of a New World monkey, the marmoset. Horizontal sections or pieces of whole retinae were double-labelled with an antiserum to S-cone opsin to identify S-cones and antibodies to the cell adhesion molecule CD15 to identify OFF-midget bipolar cells. Peanut agglutinin coupled to a fluorescent tag was used to label the cone pedicles of all cone types. Peanut agglutinin was also used to distinguish S-cones from the other cone types. The sections were analysed with deconvolution microscopy. We found that nearly all pedicles of medium- and long-wavelength-sensitive cones are located opposite distinct dendritic clusters formed by OFF-midget bipolar cells. By contrast, the S-cone pedicles are not located opposite dendritic clusters. Instead, S-cones make sparse contacts with CD15-labelled processes. Some of these processes protruded from OFF-midget bipolar clusters, whereas others could be traced to a diffuse bipolar cell type. Thus, in the marmoset retina the midget-parvocellular system does not carry a blue-OFF signal.

Animals↗

PubMed2001

Guard cell volume and pressure measured concurrently by confocal microscopy and the cell pressure probe.

Guard cell turgor pressures in epidermal peels of broad bean (Vicia faba) were measured and controlled with a pressure probe. At the same time, images of the guard cell were acquired using confocal microscopy. To obtain a clear image of guard cell volume, a fluorescent dye that labels the plasma membrane was added to the solution bathing the epidermal peel. At each pressure, 17 to 20 optical sections (each 2 microm thick) were acquired. Out-of-focus light in these images was removed using blind deconvolution, and volume was estimated using direct linear integration. As pressure was increased from as low as 0.3 MPa to as high as 5.0 MPa, guard cell volume increased in a saturating fashion. The elastic modulus was calculated from these data and was found to range from approximately 2 to 40 MPa. The data allow inference of guard cell osmotic content from stomatal aperture and facilitate accurate mechanistic modeling of epidermal water relations and stomatal functioning.

Fabaceae↗

PubMed2001

Tumor cell invasion of model 3-dimensional matrices: demonstration of migratory pathways, collagen disruption, and intercellular cooperation.

We report a novel 3-dimensional model for visualizing tumor cell migration across a nylon mesh-supported gelatin matrix. To visualize migration across these model barriers, cell proteolytic activity of the pericellular matrix was detected using Bodipy-BSA (fluorescent upon proteolysis) and DQ collagen (fluorescent upon collagenase activity). For 3-dimensional image reconstruction, multiple optical images at sequential z axis positions were deconvoluted by computer analysis. Specificity was indicated using well-known inhibitors. Using these fluorescent proteolysis markers and imaging methods, we have directly demonstrated proteolytic and collagenolytic activity during tumor cell invasion. Moreover, it is possible to visualize migratory pathways followed by tumor cells during matrix invasion. Using cells of differing invasive potentials (uPAR-negative T-47D wild-type and uPAR-positive T-47D A2--1 cells), we show that the presence of the T-47D-A2--1 cells facilitates the entry of T-47D wild-type cells into the matrix. In some cases, wild-type cells follow T-47D A2--1 cells into the matrix whereas other T-47D-wild-type cells appear to enter without the direct intervention of T-47D A2--1 cells. Thus, we have developed a new 3-dimensional model of tumor cell invasion, demonstrated protein and collagen disruption, mapped the pathways followed by tumor cells during migration through an extracellular matrix, and illustrated cross-talk among tumor cell populations during invasion.

Cell Movement↗

PubMed2002

Dynamics of tight and adherens junctions under EGTA treatment.

The dynamics of tight junctions (TJs) and adherens junctions (AJs) under EGTA treatment were investigated in Madin Darby canine kidney (MDCK) cells. Detailed information about the behavior of TJ and AJ proteins during the opening and resealing of TJs and AJs is still scarce. By means of the "calcium chelation" method, the distribution and colocalization of junctional proteins were studied with confocal laser scanning microscopy using a deconvolution algorithm for high-resolution images. Colocalization was analyzed for pairs of the following proteins: ZO-1, occludin, claudin-1, E-cadherin and F-actin. Significant differences were found for the analyzed pairs in control cells compared to EGTA-treated cells with respect to the position of the colocalization maxima within the cell monolayers as well as with respect to the amount of colocalized voxels. Under EGTA treatment, colocalization for ZO-1/occludin, ZO-1/claudin-1, claudin-1/occludin, E-cadherin/occludin and E-cadherin/claudin-1 dropped below 35% of the control value. Only for the ZO-1/E-cadherin pair, the amount of colocalized voxels increased and a shift to a more basal position was observed. During the opening of TJs and AJs, ZO-1 colocalized with E-cadherin in the lateral membrane region, whereas in controls, ZO-1 colocalized with occludin and claudin-1 in the junctional complex. The combination of deconvolution with colocalization analysis of confocal data sets offers a powerful tool to investigate the spatial relationship of TJ and AJ proteins during assembly and disassembly of cell-cell contacts.

Adherens Junctions↗

PubMed2004

The evolution of microarrayed compound screening.

This review describes recent developments in the evolutionary process of microarrayed compound screening (microARCS to become a robust and efficient ultra-high-throughput screening technology. Improvements in compound spotting (including new quality-control methods), gel casting and imaging, together with software capable of automatic analysis and deconvolution of images, have helped to streamline the screening process. A variety of screening projects using cell-based and non-cell-based approaches have been successfully concluded using microARCS. Comparison of hits derived from standard microtitre-plate-based screening and from microARCS reveals excellent overlap. Furthermore, there seems to be no bias towards finding compounds within a particular range of logP values, even though compounds are solubilized from a dry state during the course of the assay.

Biological Assay↗

PubMed1985

Cerebral blood flow in hypertensive patients with cerebrovascular disease: technique for measurement and effect of captopril.

Nineteen patients with unilateral cerebrovascular disease underwent cerebral blood flow (CBF) measurements; ten had been receiving conventional therapy and then were studied after treatment with captopril without or with a diuretic and nine on conventional therapy were studied twice as a control group. CBF (ml min-1) was measured after an intravenous injection of 99Tcm-labelled patient's red cells with a computer linked gamma camera over the vertex and a probe over the aorta. With deconvolution analysis regional CBF is given by regional cerebral volume divided by regional mean transit time. Results in the captopril group showed on average a 10% fall in mean blood pressure and a 10% rise in blood flow to the affected hemisphere. In the control group there was on average a 4% fall in pressure, together with an 11% fall in CBF to the affected hemisphere. Captopril appears to maintain autoregulation in cerebrovascular disease.

Adult↗

PubMed2001

Insulin secretion rate during glucose stimuli: alternative analyses of C-peptide data.

The ability to evaluate the pancreatic insulin secretion rate (ISR) is essential for a quantitative understanding of the glucose regulation system in man. Various approaches have been developed for evaluation of the ISR in vivo. The aim of this study was to compare input/output and compartmental models of C-peptide to reconstruct the ISR in response to both physiological and nonphysiological glucose stimuli in healthy humans. In particular we applied the nonparametric stochastic deconvolution and the C-peptide minimal model approaches to the graded up&down glucose infusion protocol, where glucose was infused at progressively increasing and then decreasing rates, and to the intravenous glucose tolerance test (IVGTT), where an impulse dose of glucose was administered. Our results show that the two models give virtually identical results when glucose and C-peptide (and thus ISR) profiles are smooth and regular, but when vigorous nonstationarities are present, like during the first 4 min of the IVGTT, the two ISR profiles are different (but not their areas under the curve). The C-peptide minimal model, albeit requiring, at variance with deconvolution, the knowledge of glucose data, has the advantage of providing quantitative indices of the beta-cell function, which is important in the parametric definition of different physiopathological states.

Adult↗

PubMed1985

An alternative to absorbed dose, quality, and RBE at low exposures.

The microdosimetric distribution of event sizes, especially for small exposures and high-LET radiation, represents both a fractional involvement of the exposed cell population and variable amounts of energy transferred to the "hit" cells. To determine the fraction of cells that will respond quantally (be transformed) after receiving a hit of a given size, a hit size effectiveness function (HSEF) which appears to have a threshold has been derived from experimental data for pink mutations in Tradescantia. The value of the HSEF at each event size, multiplied by the fractional number of cells hit at that event size, and summed over all event sizes, yields a single value representing the fractional number of quantally responding cells and thus the population impairment for a given exposure. The HSEF can be obtained by unfolding (deconvoluting) several sets of biological and microdosimetric data obtained with radiation of overlapping event size distributions.

Dose-Response Relationship, Radiation↗

PubMed1996

Deconvolved electrical impedance spectra track distinct cell morphology changes.

A two-component Cole-Cole model was used to obtain statistically significant fits to 100-Hz-10-MHz impedance data for EMT-6 mouse tumors during the progressive histological changes induced by hyperthermia. The resulting fitting parameters were used to deconvolute and reconstruct the two dispersions which confer the predominant impedance features to this tissue. The time-dependent changes of these two dispersions were correlated with the concurrent, heat-induced morphological changes of the tumors' cells. The higher frequency dispersion (fc approximately 1 MHz) was identified with a Maxwell-Wagner relaxation process linked to the overall volume response of the cells. The lower frequency dispersion (fc approximately 10 kHz) represented an alpha-relaxation associated with the surface morphology and integrity of the plasma membranes. Thus, two aspects of the characteristic cellular damage sequence in these tumors were found to be separately discernable and trackable in real-time using the impedance data.

Animals↗

PubMed1984

NMR methods for characterizing the state of the surfaces of complex mammalian cells.

It is shown that narrow 1H NMR resonances may be observed in cancer cells, and that these belong to fatty acyl chains of membrane lipids. A variety of NMR techniques such as Gaussian-Lorentzian deconvolution, and T1 and T2 measurements, may be used to subdivide these resonances further. The results of these various methods require that in the membrane structures the observed lipids tumble isotropically and sufficiently rapidly to give motionally narrowed 1H NMR lines.

Animals↗

PubMed2003

[Ca2+]i regulates trafficking of Cav1.3 (alpha1D Ca2+ channel) in insulin-secreting cells.

Chronic exposure of pancreatic beta-cells to high concentrations of glucose impairs the insulin secretory response to further glucose stimulation. This phenomenon is referred to as glucose desensitization. It has been shown that glucose desensitization is associated with abnormal elevation of beta-cell basal intracellular free Ca2+ concentration ([Ca2+]i). We have investigated the relationship between the basal intracellular free Ca2+ and the L-type (Cav1.3) Ca2+ channel translocation in insulin-secreting cells. Glucose stimulation or membrane depolarization induced a nifedipine-sensitive Ca2+ influx, which was attenuated when the basal [Ca2+]i was elevated. Using voltage-clamp techniques, we found that changing [Ca2+]i could regulate the amplitude of the Ca2+ current. This effect was attenuated by drugs that interfere with the cytoskeleton. Immunofluorescent labeling of Cav1.3 showed an increase in the cytoplasmic distribution of the channels under high [Ca2+]i conditions by deconvolution microscopy. The [Ca2+]i-dependent translocation of Cav1.3 channel was also demonstrated by Western blot analysis of biotinylation/NeutrAvidin-bead-eluted surface proteins in cells preincubated at various [Ca2+]i. These results suggest that Cav1.3 channel trafficking is involved in glucose desensitization of pancreatic beta-cells.

Animals↗

PubMed2006

Postprandial suppression of glucagon secretion depends on intact pulsatile insulin secretion: further evidence for the intraislet insulin hypothesis.

Type 2 diabetes is characterized by an approximately 60% loss of beta-cell mass, a marked defect in postprandial insulin secretion, and a failure to suppress postprandial glucagon concentrations. It is possible that postprandial hyperglucagonemia in type 2 diabetes is due to impaired postprandial insulin secretion. To address this, we studied eight adult Goettingen minipigs before and after an approximately 60% reduction in beta-cell mass induced by alloxan. Pigs were studied fasting and after ingestion of a mixed meal. Insulin and glucagon secretion were determined by deconvolution of blood hormone concentrations measured at 1-min intervals. The relationship between insulin and glucagon release was analyzed using cross-correlation and forward versus reverse cross-approximate entropy. We report that glucagon and insulin were secreted in approximately 4-min pulses. Prealloxan, postprandial insulin secretion drove an approximately 20% suppression of glucagon concentrations (P < 0.01), through inhibition of glucagon pulse mass. The alloxan-induced approximately 60% deficit in beta-cell mass lead to an approximately 70% deficit in postprandial insulin secretion and loss of the postprandial insulin-driven suppression of glucagon secretion. We conclude that postprandial hyperglucagonemia in type 2 diabetes is likely due to loss of intraislet postprandial suppression of glucagon secretion by insulin.

Animals↗