SEARCH · PubMed Health

Results for “Intracellular quantification”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 811 records · Page 45Linked to original sources

PubMed2002

Frequent downregulation of Fas (CD95) expression and function in melanoma.

Membrane-bound Fas ligand (FasL, Apo-1L, CD95L) induces rapid apoptosis of Fas (CD95)-sensitive cells on interaction with Fas, and is an important effector molecule of cytolytic T lymphocytes (CTLs). Melanomas are immunogenic and induce the production of specific CTLs, but are usually able to escape immune destruction. We investigated Fas expression and function in 53 cutaneous melanocytic lesions and 13 melanoma cell lines grown in vitro. Immunohistochemical analysis of Fas expression in cutaneous melanocytic lesions showed moderate to high levels of Fas in common benign melanocytic naevi, but low to undetectable levels in atypical naevi, primary (superficial spreading melanoma, nodular melanoma) and cutaneous melanoma metastases. Fluorescence-activated cell sorting (FACS) analysis of Fas expression in melanoma cell lines revealed undetectable or low levels of cell surface Fas expression in five of the 13 melanoma cell lines. Analysis of Fas signalling by quantification of cell death following exposure to recombinant FasL showed that a reduction in Fas expression results in resistance to FasL-mediated cell death. Furthermore, two of the 13 melanoma cell lines were found to be resistant to FasL-mediated cell death despite conserved Fas expression. Thus seven of the 13 melanoma cell lines were found to have impaired Fas signalling. Taken together, our results indicate that downregulation of Fas expression and resistance to Fas-mediated apoptosis are frequent in melanoma.

Apoptosis↗

PubMed2004

In situ imaging of mitochondrial outer-membrane pores using atomic force microscopy.

Here we describe a technique for imaging of the outer contours of the mitochondrial membrane using atomic force microscopy, subsequent to or during a toxic or metabolic challenge. Pore formation in both glucose-challenged and 1,3-dinitrobenzene (DNB)-challenged mitochondria was observed using this technique. Our approach enables quantification of individual mitochondrial membrane pore formations. With this work, we have produced some of the highest resolution images of the outer contours of the in situ mitochondrial membrane published to date. These are potentially the first images of the component protein clusters at the time of formation of the mitochondrial membrane transition pore in situ. With the current work, we have extended the application of atomic force microscopy of mitochondrial membranes to fluid imaging. We have also begun to correlate 3-D surface features of mitochondria dotted with open membrane pores with features previously viewed with electron microscopy (EM) of fixed sections.

Animals↗

PubMed2005

Albumin turnover: FcRn-mediated recycling saves as much albumin from degradation as the liver produces.

It is now understood that the nonclassical major histocompatibility complex-I molecule FcRn binds albumin and retrieves it from an intracellular degradative fate. Whether FcRn in the liver modulates albumin turnover through effects on biosynthesis and production is not known. Thus we quantified the appearance of biosynthetically labeled albumin in plasma after an intravenous bolus injection of [(3)H]leucine in FcRn-deficient mice. The production rates for both albumin (FcRn substrate) and transferrin (nonsubstrate) are increased by approximately 20% in FcRn-deficient mice compared with normal mice, likely compensating for the lowered plasma oncotic pressure caused by hypoalbuminemia in FcRn-deficient mice. Determining the magnitude of FcRn-mediated effects on albumin turnover, we then measured the steady-state plasma concentrations of biosynthetically labeled albumin and transferrin during [(3)H]leucine infusion. The concentration of albumin was approximately 40% lower in FcRn-deficient mice compared with normal mice. Furthermore, the approximately 40% lower plasma albumin concentration in FcRn-deficient mice along with the approximately 20% increase in albumin production indicate, by the mass-balance equation, that albumin degradation in FcRn-deficient mice is twice that of normal mice. These studies of biosynthetically labeled, and thus native, albumin support our previous finding that FcRn protects albumin from degradation. Permitting quantification of the magnitude of FcRn-mediated recycling, they further indicate that FcRn has extraordinary capacity: the amount of albumin saved from degradation by FcRn-mediated recycling is the same as that produced by the liver.

Albumins↗

PubMed1988

Bromodeoxyuridine enhancement of 1-beta-D-arabinofuranosylcytosine metabolic activation and toxicity in HL-60 leukemic cells.

We tested whether bromodeoxyuridine (BrdUrd), an analogue of thymidine (dThd), enhances 1-beta-D-arabinofuranosylcytosine (ara-C) metabolic activation, as does dThd. HL-60 cells were exposed to 10, 100, or 1000 nM ara-C for 3 h. Simultaneous exposure of log phase HL-60 cells to BrdUrd (1-1000 microM) and ara-C for 3 h resulted in enhancement of ara-C incorporation into DNA, with a doubling of incorporation in response to 10 nM ara-C occurring at concentrations of BrdUrd greater than 100 microM. Preexposure of cells to BrdUrd for 16 h followed by addition of ara-C for 3 h resulted in even greater ara-C incorporation into DNA. This increase was most marked at the lower concentrations of ara-C (10 and 100 nM), where approximately 3-fold enhancement of ara-C incorporation was observed in response to BrdUrd concentrations greater than 100 microM. Intracellular pools of 1-beta-D-arabinofuranosyl-CTP increased significantly (up to 3-fold) following 16-h exposure to BrdUrd (30, 100, or 300 microM) at all concentrations of ara-C tested. The ara-C phosphorylating activity of cell-free extracts obtained following 16-h exposure of cells to BrdUrd increased 1.5- to 2.3-fold over control. Intracellular dCTP pools fell to approximately 50% of control after exposure to 750 microM BrdUrd or dThd. Exposure to BrdUrd for 16 h caused a concentration-dependent increase in cells with S-phase DNA content, as assessed by flow cytometry, with a doubling of cells in S phase (to 60%) observed in response to 500 microM BrdUrd. HL-60 cells exposed to identical conditions of BrdUrd for 3 h showed no significant alteration in cell cycle phase distribution. Thus, although BrdUrd does increase cells in S phase, the increased ara-C incorporation caused by BrdUrd cannot be explained solely on a cytokinetic basis since enhancement of incorporation was observed after a 3-h exposure of cells to BrdUrd and ara-C. The combination of ara-C (100 nM) and BrdUrd (100-1000 microM) exhibited cytotoxic synergism, as measured by the fluorescein diacetate/propidium iodide method. These data demonstrate a clear potential for BrdUrd modulation of ara-C metabolism in human leukemia. Additionally, the interaction of BrdUrd and ara-C should be considered in the interpretation of studies of the effects of ara-C on DNA synthesis as measured by flow cytometric quantification of incorporated BrdUrd.

Arabinofuranosylcytosine Triphosphate↗

PubMed1991

Production of antisera selective for m1 muscarinic receptors using fusion proteins: distribution of m1 receptors in rat brain.

A fragment of the cDNA encoding the third intracellular loop of the rat m1 muscarinic receptor was cloned, and the DNA was expressed in Escherichia coli as a fusion protein. The fusion protein was purified and utilized as an antigen to raise a polyclonal antiserum in rabbits. Chinese hamster ovary cells stably transfected with the cDNA encoding each of the five known subtypes of muscarinic receptor were used as tissue sources to test the antiserum. The antiserum was found to quantitatively immunoprecipitate m1 muscarinic receptors, while not precipitating m2, m3, m4, or m5 receptors. This selective antiserum was utilized to quantify the density of m1 muscarinic receptors in seven selected areas of the rat brain. Thus, cortex was found to contain approximately 0.8 pmol/mg of membrane protein, which represents 34% of the total density of muscarinic receptors. Similarly, hippocampus (1 pmol/mg; 47%), striatum (0.8 pmol/mg; 29%), and olfactory tubercule (0.9 pmol/mg; 35%) are rich in m1 receptors. In contrast, thalamus/hypothalamus contained only 0.15 pmol/mg, representing approximately 16% of the total density of muscarinic receptors, whereas pons/medulla (0.03 pmol/mg; 5%) and cerebellum (less than 0.01 pmol/mg; 2%) had very low levels of expression of m1 receptors. The development of a selective antiserum has provided a means for the quantification of a specific subtype of muscarinic receptor in tissues, such as the brain, that express multiple subtypes. This methodology will be applicable not only to the other subtypes of muscarinic receptor but also to the subtypes of several other neurotransmitter receptors that lack selective drugs with which to study them.

Animals↗

PubMed1983

Induction and repression of the major phenobarbital-induced cytochrome P-450 measured by radioimmunoassay.

Two independent radioimmunoassay techniques for the major phenobarbital-inducible cytochrome P-450 (PB P-450) of rat liver microsomal membranes are described. The first technique employs as the source of radiolabelled antigen the products of translation in vitro labelled with [35S]methionine. The second technique employs purified antigen labelled with 125I and is quicker, less expensive and more precise. Both assays are highly specific for PB P-450 and can detect quantities of this variant as small as 1 ng. This is several orders of magnitude more sensitive than any method described previously for the quantification of cytochromes P-450, and consequently the technique is particularly well suited for the quantification of so-called constitutive cytochrome P-450 variants that are present in very low amounts. The results of the radioimmunoassays demonstrate that the apparent 2.6-fold induction of total cytochromes P-450 after phenobarbital treatment is due to a 43-fold increase in Pb P-450. Although beta-naphthoflavone increases the total content of cytochrome P-450 of microsomal membranes 1.4-fold, it actually causes a 55% decrease in the amount of PB P-450. Thus different xenobiotics can have differential effects on the expression of the genes for specific cytochrome P-450 variants.

Animals↗

PubMed1977

Biophysical mechanisms of anesthetic action: historical perspective and review of current concepts.

The large number and diversity of anesthetic agents were evident to investigators 80 years ago, and suggested a physicochemical theory of anesthesia. Meyer and Overton were the first to offer a quantitative relationship between a physicochemical property and potency of anesthetic agents. They also focused attention on the lipid phase as the site of anesthetic action. Ferguson realized that the concentration of an agent at its site of action bears a generally unknown relation to the concentration in the external phase. However, at equilibrium the activity of an agent is the same in every phase, motivating Ferguson to suggest that activities rather than concentrations be used as indices of dosage. The critical-volume theory resulted from modification of the Meyer-Overton theory to include the molal volume of the anesthetic. The allowance for molal volume resulted initially from an attempt further to regularize the experimental data. The concept of a critical-volume fraction of anesthetic being necessary for narcosis was discussed in most detail by Mullins. Subsequently, the concept of the effect of the anesthetic has changed from filling of free space to expansion and fluidization of the membrane. The ability of pressure to cause excitant phenomena and antagonize anesthetics is predictable from the critical-volume theory and is therefore highly significant evidence. K. W. Miller and associates are perhaps most prominent in the recent quantification and formalization of the critical-volume theory and HPNS. The existence of a separate convulsant site(s) is suggested by the demonstration of significantly different compressibilities associated with anesthesia and convulsions. Work corroborating a separate convulsant site involved measurement of the partial molal volumes of a series of related convulsant and anesthetic ethers and calculation of each compound's solubility parameter. Multiple convulsant sites may exist, and these two methods may not have accessed the same site. Understanding the anesthetic-convulsant duality will have important practical application to deepwater diving, and may well offer important insight into the neurophysiologic and electrophysiologic effects of anesthetics. The application of ESR and NMR allows investigation at the molecular level of effects of anesthetics on biological and model membranes. Magnetic resonance techniques have generally supported the concept of membrane fluidization by anesthetics. Some investigators have recently attempted to displace the focus of attention from the lipid phase. However, the evidence is clearly against the aqueous-phase theory of Pauling and S.L. Miller. The microtubule theory of Allison and Nunn has not accumulated supporting evidence comparable to the lipid theories. Contradictory evidence makes any evaluation of this theory speculative. Additionally, the interspecies and intracellular variability of microtubules raises questions of the relevance of many studies...

Anesthesia, General↗

PubMed2006

A specific picomolar hybridization-based ELISA assay for the determination of phosphorothioate oligonucleotides in plasma and cellular matrices.

PURPOSE: To develop and validate an ultrasensitive and specific hybridization-based enzyme-linked immunosorbent assay method for quantification of two phosphorothioate oligonucleotides (PS ODNs) (G3139 and GTI-2040) in biological fluids. METHODS: This assay was based on hybridization of analytes to the biotin-labeled capture ODNs followed by ligation with digoxigenin-labeled detection ODN. The bound duplex was then detected by anti-digoxigenin-alkaline phosphatase using Attophos (Promega, Madison, WI, USA) as substrate. S1 nuclease and major factors such as the hybridization temperature, concentration of capture probe, and the use of detergent were evaluated toward assay sensitivity, selectivity, and accuracy. RESULTS: The method is selective to the parent drugs with minimal cross-reactivity (<6%) with 3'-end deletion oligomers for both G3139 and GTI-2040. A linear range of 0.05 to 10 nM (r2 > 0.99) was observed for GTI-2040 in a variety of biological matrices. For both G3139 and GTI-2040, the within-day precision and accuracy values were found to be <20% and 90-110%, respectively; the between-day precision and accuracy were determined to be <20% and 90-120%. Addition of S1 nuclease combined with washing step greatly improved the assay linearity and selectivity. The utility of this assay was demonstrated by simultaneous determination of GTI-2040 in plasma and its intracellular levels in treated acute myeloid leukemia patients. CONCLUSIONS: The validated hybridization enzyme-linked immunosorbent assay method is specific for quantitation of PS ODNs in biological samples to picomolar level. This method provides a powerful technique to evaluate plasma pharmacokinetics and intracellular uptake of PS ODNs in patients and shows its utility in clinical evaluations.

Acute Disease↗

PubMed1999

Assessing the mechanism of metabolism-dependent valproic acid-induced in vitro cytotoxicity.

This study was designed to distinguish and evaluate the contribution of reactive metabolite and reactive oxygen species as the mechanism of metabolism-dependent valproic acid-induced in vitro cytotoxicity. The involvement of reactive oxygen species in the mechanism of in vitro cytotoxicity was examined by the addition of a series of antioxidant enzymes and iron chelators to the reaction mixture. Addition of catalase to the reaction mixture resulted in a complete prevention of valproic acid-induced cytotoxicity. Co-incubation of a cell impermeable iron chelator deferoxamine did not effect cytotoxicity, whereas 1,10-phenanthroline, a chelator with the ability to traverse cell membranes at low concentrations, afforded significant protection against valproic acid-induced cytotoxicity. A possible inhibitory effect of catalase and 1,10-phenanthroline on the microsomal metabolism of valproic acid was disproved by the quantification of valproic acid metabolites in the presence and absence of these compounds. To assess the specificity of the mechanism of in vitro valproic acid-induced cytotoxicity, prevention of in vitro acetaminophen-induced cytotoxicity by antioxidant enzymes and iron chelators was also evaluated. Addition of catalase to the reaction mixture in the presence of acetaminophen resulted in a moderate reduction in the level of but a lack of complete protection of cytotoxicity. Addition of 1,10-phenanthroline to the reaction mixture in the presence of acetaminophen did not result in a detectable change in acetaminophen-induced cytotoxicity. These data suggest the involvement of reactive oxygen species in the mechanism of toxicity of valproic acid and perhaps reactive metabolites as the major cause of cytotoxicity in the case of acetaminophen in the in vitro model investigated. Inhibition of poly(ADP-ribose) polymerase activity by various antagonists resulted in complete prevention of valproic acid-induced in vitro cytotoxicity. The cytoprotective effects of known poly(ADP-ribose) polymerase antagonists implicate poly(ADP-ribose) polymerase in the mechanism of in vitro metabolism-dependent valproic acid-induced cytotoxicity under these conditions. These results further point to nuclear DNA as the intracellular site of insult by the generated oxygen radicals. Overall, the data obtained support the hypothesis that the metabolism-dependent valproic acid-induced in vitro cytotoxicity is the result of generation of hydrogen peroxide in the medium that can readily cross cell membranes and subsequently interact intracellularly with iron to produce the highly reactive hydroxyl free radicals.

Acetaminophen↗

PubMed2003

Dynamic changes in the expression of protein tyrosine phosphatases during preimplantation mouse development: semi-quantification by real-time PCR.

Protein tyrosine phosphatase (PTP) expression was examined in preimplantation mouse embryos. We previously reported that SHP-2, LAR, PTPT9, SHP-1, and mRPTPB were expressed in preimplantation mouse embryos. Here, we examined changes in the expression levels of these PTPs during preimplantation development. cDNA was obtained by reverse transcription of embryo mRNA, amplified with 10 PCR cycles, and then subjected to real-time fluorescence-monitored PCR. Experiments with an mRNA dilution series revealed that the data obtained matched the quantities of mRNA used. The measurements obtained with real-time fluorescence-monitored PCR showed that the expression of each PTP mRNA changed dynamically, and that each had a different expression pattern. This suggests that PTPs are involved in the regulation of growth and differentiation during preimplantation development.

Animals↗

PubMed1998

Tumor necrosis factor-alpha acutely inhibits insulin signaling in human adipocytes: implication of the p80 tumor necrosis factor receptor.

Tumor necrosis factor (TNF)-alpha is postulated to play a major role in the pathogenesis of obesity-linked insulin resistance, probably resulting from an interaction with insulin signaling pathways. This cross talk has now been investigated in human adipocytes at the level of phosphatidylinositol (PI) 3-kinase, and the TNF receptors (TNFRs) mediating these processes have been identified. Equilibrium binding studies using human adipocytes from mammary tissue indicated the presence of two populations of TNFR with apparent affinity constants of 13 pmol/l and 1.6 nmol/l, respectively. Interaction of TNF-alpha with insulin signaling was determined by quantification of insulin receptor substrate (IRS)-1-associated PI 3-kinase activity. Under control conditions, PI 3-kinase was activated about 10-fold in response to insulin (10[-7] mol/l, 5 min). Preincubation of adipocytes with 5 nmol/l TNF-alpha for 15 min resulted in a 60-70% reduction of insulin action, reaching a stable inhibition (40%) after longer incubation with the cytokine. The inhibitory action of TNF-alpha was dose-dependent, already detectable at 10 pmol/l, and was correlated to inhibition of tyrosine phosphorylation of IRS-1 with an unaltered autophosphorylation of the insulin receptor beta-subunit. The modulation of insulin signaling by TNF-alpha was found to be paralleled by a comparable inhibition of insulin-stimulated glucose transport. An agonistic TNFR1 antibody completely mimicked the inhibitory action of TNF-alpha on insulin signaling, whereas at 100 pmol/l TNF-alpha, a nonagonistic p80 TNFR antibody, was shown to ameliorate the inhibitory action of the cytokine. These findings indicate that in human adipocytes, low concentrations of TNF-alpha induce a rapid inhibition of insulin signaling at the level of PI 3-kinase. We suggest that under these conditions, the p80 TNFR is essential for initiating the intracellular cross talk that involves signaling by the p60 TNFR.

Adipocytes↗

PubMed2000

Quantification of relative mRNA expression in the rat brain using simple RT-PCR and ethidium bromide staining.

We developed a protocol for quantification of relative gene expression using reverse transcription-polymerase chain reaction (RT-PCR) without the use of radioisotopes, special equipment or extra nucleotide fragments, such as competitors. The relative gene expression of GABA(A) receptor beta(1) subunit (GABA(A)Rbeta(1)) and phospholipase C beta(4) subtype (PLCbeta(4)) in rat cerebrum and cerebellum were determined by comparing the ratio of PCR products generated by linear amplification of the target cDNA segments and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA segment as a reference. The density of PCR products was measured from digitized images of photographs of ethidium-bromide-stained agarose gels. The linear region of PCR amplification was within the linear range (from 0.3 to 12 ng DNA in a single band) of the detection system. The accuracy of the present method was <2-fold difference in gene expression in a single determination and a 1.5-fold difference was statistically significant after repeated measurements. The estimated relative expression of PLCbeta(4) was significantly higher in cerebellum than cerebrum, and that of GABA(A)Rbeta(1) was the same in these two regions. Using the present method, it is possible to quantify several different subunits and subtypes of known ion channel, neurotransmitter receptor and intracellular signaling enzyme gene families.

Animals↗

PubMed1987

Plasma tetranectin in healthy male and female individuals, measured by enzyme-linked immunosorbent assay.

Tetranectin is a novel protein recently isolated from human plasma. It is a tetramer, composed of four identical, non-covalently bound, 181-amino-acid polypeptide chains (Mr = 20,100). We report here the quantification of plasma tetranectin in 457 healthy individuals, aged from birth to 85 years, using a newly developed sensitive and reproducible enzyme-linked immunosorbent assay (ELISA). Tetranectin was demonstrable in all subjects investigated, and within each sex and defined age group the concentration was well controlled within a relatively narrow range. The mean plasma tetranectin level in newborn infants (cord plasma) was about 8 mg/L. This was significantly less than in later life, during which mean plasma tetranectin level varied between 10 and 12 mg/L. After the age of 9 years, tetranectin level was continuously higher in males than in females, but the variations through the span of life were almost identical. Thus, a transitory increase in plasma tetranectin was observed in early puberty, reaching its climax about the age of 11 to 12 in girls and 14 to 15 in boys. A quantitatively similar, additional peak was observed during the period of 50 to 59 years of age in both sexes, whereupon the tetranectin concentration gradually decreased. The biologic function of tetranectin remains to be elucidated. Recently, we have reported that tetranectin is contained within hepatocytes and various endocrine cells, all known to process peptide hormones or glucoproteins. We propose that tetranectin may be involved in intracellular and extracellular serine protease-mediated proteolysis.

Adolescent↗

PubMed1997

Stereological image analysis of cultured human melanocytes observed by transmission electron microscopy.

The aims of the study were to write an image analysis (IA) program allowing the stereological quantification of human epidermal melanocyte melanization at the ultrastructural level and to specify the suitable preparative methods, in keeping with IA limits and stereological principles. Micrographs of cultured human melanocytes obtained in transmission electron microscopy were digitized with a scanner. The key step of the designed IA program is a thresholding based on the gray levels. Hence, gray level histograms (pixel frequency as a function of gray level) of melanocyte images exhibit a peak specific to melanin. The gray level thresholding used consists in isolating the melanin pixels that form profiles on a binary image and in storing the numerical data produced for a given melanocyte profile. These primary data are used to calculate numerous parameters via stereology with melanocyte cytoplasm and melanized melanosome as main reference spaces. The most important stereological parameters obtained are v(mi,cy) (melanin volume per average cell), v(mi,m) (melanin volume per average melanized melanosome), and nm (number of melanized melanosomes per average cell), and their validity is discussed. Melanocytes embedded in situ were abandoned for stereological reasons but pelleted melanocytes were found suitable. Using this computerized tool and stereology, we are able to perform quantitative studies producing varied data even from small cell samples. To our knowledge, this is the first stereological approach for quantifying intracellular melanization. A quantitative comparison of spectrophotometrical results (melanin assay) with stereological results obtained in ultraviolet B-irradiated Caucasian epidermal melanocytes will be performed in order to appraise this method.

Cytoplasm↗

PubMed1996

Inhibition of glutathione S-transferase activity in human melanoma cells by alpha,beta-unsaturated carbonyl derivatives. Effects of acrolein, cinnamaldehyde, citral, crotonaldehyde, curcumin, ethacrynic acid, and trans-2-hexenal.

The glutathione S-transferase (GST) activity towards 1-chloro-2,4-dinitrobenzene in intact human IGR-39 melanoma cells was determined by the quantification by HPLC-analysis of the excreted glutathione (GSH) conjugate (S-(2,4-dinitrophenyl)glutathione; DNPSG). The major GST subunit expressed in these melanoma cells is the pi-class GST subunit P1. Using this system, the effect of exposure for 1 h to a series of alpha, beta-unsaturated carbonyl compounds at non-toxic concentrations was studied. Curcumin was the most potent inhibitor (96% inhibition at 25 microM), while 67 and 61% inhibition at 25 microM was observed for ethacrynic acid and trans-2-hexenal, respectively. Moderate inhibition was observed for cinnamaldehyde and crotonaldehyde, while no inhibition was found for citral. The reactive acrolein did not inhibit the DNPSG-excretion at 2.5 microM, the highest non-toxic concentration. Up to about 50% GSH-depletion was found after treatment with crotonaldehyde, curcumin and ethacrynic acid, however the consequences for GST conjugation are presumably small. Reversible inhibition of GST was the major mechanism of inhibition of DNPSG-excretion in melanoma cells, except in the cases of curcumin and ethacrynic acid, which compounds also inactivated GSTP1-1 by covalent modification. This was clear from the fact that depending on the dose between 30 and 80% inhibition was still observed after lysis of the cells, under which conditions reversible inhibition was is absent. Intracellular levels of DNPSG remained relatively high in the case of ethacrynic acid. It is possible that ethacrynic acid also inhibits the transport of DNPSG by inhibition of the multidrug resistance-associated protein gene encoding glutathione conjugate export pump (MRP/GS-X pump) in some way.

Acrolein↗

PubMed1989

In-vivo quantification of myocardial Na-K pump rate during beta-adrenergic stimulation of intact pig hearts.

Maintenance of adequate electrical activity of the heart depends critically on the ability of the Na-K pump to compensate for normal passive sodium and potassium fluxes. Using sudden injections of [3H]ouabain into the left coronary artery in anaesthetized open-chest pigs, we monitored transient changes in myocardial potassium balance by PVC-valinomycin mini-electrodes. When related to the number of pumps blocked and fractional inhibition, these data provided estimates of total Na-K pump capacity as well as actual pump rate and perturbations of the Na-K balance. Experiments were performed in hearts with and without intracoronary isoprenaline infusion (2.5 nmol min-1). After injection of 120 nmol [3H]ouabain into the left coronary artery, myocardial [3H]ouabain concentrations were 118 (74-178) and 103 (76-145) pmol g-1 and total concentrations of [3H]ouabain binding sites were 893 (752-1076) and 785 (691-877) pmol g-1 (median, 95% confidence interval) in isoprenaline-treated and control hearts respectively (differences not significant). The [3H]ouabain injection caused a net potassium release of 81 (56-132) and 43 (23-75) mumol 100 g-1 (median, 95% confidence interval) in isoprenaline-treated and control hearts respectively (n = 6-8; significance of difference, P = 0.03). Na-K pump rate estimated from mono-exponential release curves was 6363 (3942-10,858) K+ ions min-1 site-1 during beta-adrenoceptor stimulation and 2514 (1380-4322) in control (significance of difference, P = 0.03). This corresponds to 40 and 16%, respectively, of the maximum possible pump rate determined from ATP hydrolysis. Comparison of accumulated potassium release and relative Na-K pump rate indicates that catecholamines enhance the sensitivity of the Na-K pump for intracellular sodium.

Adrenergic beta-Agonists↗

PubMed1990

Increased expression of high-affinity low-density lipoprotein receptors on human T-blasts.

Like all cells, lymphocytes need cholesterol for proper function, a requirement met by a finely tuned homeostasis between intracellular synthesis and uptake from the environment via low-density lipoproteins (LDL). We used flow cytometry to analyze the receptor activity of resting cells and T blasts incubated/activated in serum-free culture medium, or in medium supplemented with 25-5,000 micrograms/ml LDL. Dioctadecyl-indocarbocyanine has proved to be a useful fluorescent probe for investigating the LDL receptor activity of lymphocytes. The results show the receptor activity of day-3 resting T cells to be reduced more than 50% by 50 microgram LDL/ml, whereas 100-fold higher concentrations are necessary to achieve the same level of reduction in day-3 PHA blasts. The LDL receptor activities of individual blood donors' resting T cells, in vitro cholesterol-deprived resting T cells, and activated T blasts, were compared using two analytical techniques: spectrofluorometric analysis of detergent-solubilized cell suspensions and flow cytometric analysis of single living cells. Receptor affinity was determined by Scatchard analysis of spectrofluorometric binding curves, and by Line-weaver-Burke plots of flow cytometric data. Both methods yielded essentially identical dissociation constants (Kd) for cholesterol-deprived resting T cells and mitogen-activated T blasts, which fell in the expected range for the high-affinity LDL receptor (4.1-8.9 nM). In addition, spectrofluorometric analysis, but not flow cytometry, permitted quantification of LDL uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1989

Molecular interactions of cultured turkey kidney cells with specific antigens of Eimeria adenoeides sporozoites.

Earlier studies suggested that specific communication between the parasite and the host cell may play a role in cellular invasion by sporozoites of species of avian Eimeria. In this study, quantification of cellular invasion and modified Western blot analysis were used to explore the possibility that parasite receptors for interaction with the host cell might be involved in the sporozoite-host cell communication. Invasion in cultured cells treated with a homogenate of Eimeria adenoeides sporozoites was approximately 50% lower than that in untreated cultures. When the sporozoite homogenate was solubilized in sodium dodecyl sulfate and electrophoretically separated, components of the cultured host cells bound consistently to sporozoite bands having Mr of 23 and 40 kDa. Biotinylation of intact sporozoites revealed at least 14 biotin-labeled bands, including bands at 23 and 40 kDa, that were considered to be surface molecules. If the sporozoites were incubated in trypsin after they were biotinylated, only two biotinylated bands at 18 and 23 kDa remained; the 40-kDa biotinylated band was not detected. Despite the removal of the majority of the surface molecules, the cell homogenate still bound to the trypsin-treated sporozoites; the intensity of the label was similar to that resulting from the binding of cell homogenate to untreated sporozoites. The data show specific interactions between 23- and 40-kDa sporozoite bands and host cell components, and provide evidence that the 23-kDa molecule may be located on the sporozoite surface and the 40-kDa molecule located intracellularly.

Animals↗