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Results for “Intracellular quantification”

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PubMed1995

Direct evaluation of intracellular accumulation of free and polymer-bound anthracyclines.

Nanoparticulate carriers of anthracyclines are being developed with the aim of improving the pharmacokinetic or pharmacodynamic behavior of these drugs. To understand how the drug reaches its nuclear targets, we have developed two methods that allow the quantification of the interaction between an anthracycline and cellular DNA: (1) by direct evaluation of the quenching of anthracycline fluorescence due to the intercalation of the drug into DNA and (2) by the measurement of Hoechst 33258 fluorescence associated with its displacement from DNA-binding sites for which it competes with the anthracycline. We show that the intracellular accumulation and DNA binding of doxorubicin encapsulated in polyisohexylcyanoacrylate nanospheres (dox-NS) and of daunorubicin bound to polyglutamic acid are reduced by 30%-40% in comparison with those obtained for free doxorubicin (dox) and daunorubicin, respectively. The results obtained with dox or NS-dox are not modified by prior incubation with either of these compounds. The two methods yielded similar results, and we conclude that either technique is applicable to the evaluation of the interaction of carrier-bound anthracyclines with cellular DNA.

Animals↗

PubMed2001

Calcium-dependent interleukin-8 gene expression in T84 human colonic epithelial cells.

OBJECTIVE AND DESIGN: IL-8 is a chemokine that activates and recruits neutrophils and plays a major role in intestinal inflammation. Signal transduction pathways mediated by protein kinases are central in regulating IL-8 gene expression, however, little is known about the role of Ca2+ in this event. In this study, we characterize the effect of intracellular Ca2+ on interleukin-8 gene expression in T84 human colonic epithelial cells. MATERIALS AND METHODS: Cells were stimulated with Ca2+ ionophore, A23187 or thapsigargin, a Ca2+-ATPase inhibitor. Semi-quantitative RT-PCR was used to examine IL-8 mRNA and ELISA for protein quantification. Reporter gene techniques were used to determine transcription rate. RESULTS: A23187 and thapsigargin caused a dose- and time-dependent accumulation of IL-8 mRNA and protein production which was dependent on the release of Ca2+ from intracellular stores. FK506, a specific inhibitor of calcineurin, inhibited A23187- and thapsigargin-induced IL-8 mRNA expression in a dose dependent manner. Reporter gene studies and actinomycin D chase experiments showed that A23187 and thapsigargin enhanced IL-8 gene transcription and stabilized IL-8 mRNA transcripts, respectively. CONCLUSION: Intracellular Ca2+ plays an important role in regulating IL-8 transcriptionally and posttranscriptionally through calcium/calmodulin-dependent calcineurin.

Adenocarcinoma↗

PubMed2007

Agonist-dependent internalization of D2 receptors: Imaging quantification by confocal microscopy.

In positron emission tomography and single photon emission computed tomography studies using D2 dopamine (DA) receptor radiotracers, a decrease in radiotracer binding potential (BP) is usually interpreted in terms of increased competition with synaptic DA. However, some data suggest that this signal may also reflect agonist (DA)-induced increases in D2 receptor (D2R) internalization, a process which would presumably also decrease the population of receptors available for binding to hydrophilic radioligands. To advance interpretation of alterations in D2 radiotracer BP, direct methods of assessment of D2R internalization are required. Here, we describe a confocal microscopy-based approach for the quantification of agonist-dependent receptor internalization. The method relies upon double-labeling of the receptors with antibodies directed against intracellular as well as extracellular epitopes. Following agonist stimulation, DA D2R internalization was quantified by differentiating, in optical cell sections, the signal due to the staining of the extracellular from intracellular epitopes of D2Rs. Receptor internalization was increased in the presence of the D2 agonists DA and bromocriptine, but not the D1 agonist SKF38393. Pretreatment with either the D2 antagonist sulpiride, or inhibitors of internalization (phenylarsine oxide and high molarity sucrose), blocked D2-agonist induced receptor internalization, thus validating this method in vitro. This approach therefore provides a direct and streamlined methodology for investigating the pharmacological and mechanistic aspects of D2R internalization, and should inform the interpretation of results from in vivo receptor imaging studies.

Animals↗

PubMed2004

Monoclonal antibody analysis of Perkinsus marinus extracellular products.

The protozoan oyster parasite Perkinsus marinus releases a complex set of extracellular products (ECP) during in vitro culture. These products have been previously implicated in parasite virulence, and their expression can be altered by medium supplementation with oyster tissue homogenate. Little is known regarding ECP function, regulation, or mechanism of storage and release. Perkinsus marinus ECP were purified from a protein-free medium and used to produce a panel of five monoclonal antibodies. Several of the antibodies recognised series of proteins implying that the ECP may originate from comparatively few parental molecules. The ECP are secreted by several pathways, including the release of one product from an external cell layer, and two other products from two morphologically distinct intracellular compartments. Antibodies against separate epitopes on one protein provided information about possible protein structure. A sandwich ELISA format allowed sensitive quantification of that protein and showed significantly reduced protein expression in oyster tissue homogenate supplemented cultures. Immunopurification allowed tandem mass spectroscopic amino acid sequencing of that protein. Another antibody was used to characterise the P. marinus cell wall. This antibody specifically bound to trophozoite and tomont walls, and was used to investigate the morphological and antigenic changes in these walls during Ray's fluid thioglycollate medium-induced formation of hypnospores. It was also used to confirm that oyster tissue homogenate supplementation could induce formation of hypnospores. This antibody labeled P. marinus cells in fixed oyster tissue in a species-specific manner.

Amino Acid Sequence↗

PubMed1999

The characterization and quantification of antigen-induced Ca2+ oscillations in a rat basophilic leukaemia cell line (RBL-2H3).

Using the ratiometric Ca2+ indicator, indo-1, the antigen-induced increase in intracellular Ca2+ concentration ([Ca2+]i) was measured in individual RBL-2H3 cells which had been passively sensitized with monoclonal antibody to the dintrophenyl (DNP) haptenic group. Antigenic stimulation using DNP-human serum albumin conjugate (DNP-HSA) induced concentration-dependent asynchronous Ca2+ oscillations, or irregular spikes. To achieve a quantitative comparison of the effects of different concentrations of antigen on changes in Ca2+[i, the area under the curve (AUC) of Ca2+ oscillations in each cell was calculated. The dose-response curve of the calculated AUC is consistent with the bell-shaped dose-response curve for antigen-induced mediator release, depolarization and 86Rb(+)-efflux. Ca2+ oscillations induced by antigenic stimulation were abolished by removal of external Ca2+ and the subsequent reintroduction of external Ca2+ caused their resumption. To investigate the role of Ca2+ oscillations in the secretory response, changes in [Ca2+]i induced by concanavalin A (Con-A), A23187, thapsigargin and NECA were also monitored. Con-A mimicked the response induced by antigen, whilst A23187 and thapsigargin induced a large transient non-oscillatory response. NECA, an adenosine receptor agonist, induced only a small transient rise in Ca2+[i without oscillatory behaviour. Since all these stimuli accept NECA-induced degranulation in these cells, it is suggested that, although Ca2+ oscillations are not essential for the initiation of secretion, they probably underlie the in-vivo physiological response of mast cells and basophils to an antigenic challenge. They also seem to enhance the efficacy of the Ca2+ signal.

Adenosine-5'-(N-ethylcarboxamide)↗

PubMed1995

Measurement of cytosolic Ca2+ concentration in Limulus ventral photoreceptors using fluorescent dyes.

Several Ca-sensitive fluorescent dyes (fura-2, mag-fura-2 and Calcium Green-5N) were used to measure intracellular calcium ion concentration, Cai, accompanying light-induced excitation of Limulus ventral nerve photoreceptors. A ratiometric procedure was developed for quantification of Calcium Green-5N fluorescence. A mixture of Calcium Green-5N and a Ca-insensitive dye, ANTS, was injected in the cell and the fluorescence intensities of both dyes were used to calculate the spatial average of Cai within the light-sensitive R lobe of the photoreceptor. In dark-adapted photoreceptors, the initial Cai was 0.40 +/- 0.22 microM (SD, n = 7) as measured with fura-2. Cai peaked in the light-sensitive R lobe at 700-900 ms after the onset of an intense measuring light step, when the spatial average of Cai within the R lobe reached 68 +/- 14 and 62 +/- 37 microM (SD, n = 5) as measured with mag-fura-2 and Calcium Green-5N, respectively. The rate of Cai rise was calculated to be approximately 350 microM/s under the measuring conditions. The resting level of Mg2+ was estimated to be 1.9 +/- 0.9 mM, calculated from mag-fura-2 measurements. To investigate the effect of adapting light on the initial Cai level in the R lobe, a 1-min step of 420 nm background light was applied before each measurement. The first significant (P < 0.05) change in the initial level of Cai occurred even at the lowest adapting light intensity, which delivered approximately 3 x 10(3) effective photons/s. The relative sensitivity of the light-adapted photoreceptors was linearly related to the relative Cai on a double log plot with slope between -4.3 and -5.3. We were unable to detect a Cai rise preceding the light-activated receptor potential. The Cai rise, measured with Calcium Green-5N, lagged 14 +/- 5 ms (SD, n = 32) behind the onset of the receptor potential at room temperature in normal ASW. In the absence of extracellular Ca2+ and at 10 degrees C, this lag increased to 44 +/- 12 ms (SD, n = 17).

Animals↗

PubMed1998

Ajoene, a compound of garlic, induces apoptosis in human promyeloleukemic cells, accompanied by generation of reactive oxygen species and activation of nuclear factor kappaB.

The pharmacological role of garlic in prevention and treatment of cancer has received increasing attention, but thorough investigations into the molecular mechanisms of action of garlic compounds are rare. The present study demonstrates that ajoene, a major compound of garlic induces apoptosis in human leukemic cells, but not in peripheral mononuclear blood cells of healthy donors. The effect was dose and time dependent. Apoptosis was judged by three criteria, morphology of cells, quantification of subdiploid DNA content by flow cytometry, and detection of DNA fragmentation by gel electrophoresis. Ajoene increased the production of intracellular peroxide in a dose- and time-dependent fashion, which could be partially blocked by preincubation of the human leukemic cells with the antioxidant N-acetylcysteine. Interestingly, N-acetylcysteine-treated cells showed a 50% loss of ajoene-induced apoptosis. Moreover, ajoene was demonstrated to activate nuclear translocation of the transcription factor nuclear factor kappaB, an effect that was abrogated in N-acetylcysteine-loaded cells. These results suggested that ajoene might induce apoptosis in human leukemic cells via stimulation of peroxide production and activation of nuclear factor kappaB. This is a novel aspect in the biological profile of this garlic compound and an important step in elucidating the underlying molecular mechanisms of its antitumor action.

Antineoplastic Agents↗

PubMed1994

Phagocytosis of viable Candida albicans by alveolar macrophages: flow cytometric quantification.

The phagocytic capacity of blood leukocytes may be assessed by flow cytometric techniques using fluorochrome-labeled particles including viable microorganisms. Application of this approach to alveolar macrophages (AM) is hampered or even rendered impossible by the strong autofluorescence of this cell type, superimposing the fluorescence intensity of the labeled phagocytic targets. Viable Candida albicans were loaded with the membrane-permeable fluorescent dye carboxy-seminaphtorhodafluor 2/acetoxymethylester (carboxy-SNARF 2-AM), which is cleaved intracellularly to generate the membrane-impermeable derivative carboxy-SNARF 2. Fluorescence was excited with the 488-nm line of an argon-ion laser, and the emission peak at 633 nm was used for quantification of dye-associated fluorescence. Rabbit and human AM were labeled with fluorescein isothiocyanate-coupled monoclonal mouse anti-macrophage antibodies. After coincubation of macrophages and yeast, 4% paraformaldehyde plus 0.5% EDTA in phosphate-buffered saline was used to stop the phagocytic process and detach adherent yeast from the AM surface. Macrophages loaded with yeast displayed a shift from monochromatic (green) to dual (green and red) fluorescence. The percentage of yeast-positive AM and red fluorescence intensity of phagocytosing macrophages were quantified. Yeast opsonization with serum or anti-Candida immunoglobulins was a prerequisite for phagocytosis. Under optimized conditions (0.5-10% serum; 60 min yeast-AM incubation; yeast-AM ratio 8:1 to 12:1), 71-91% of the AM were involved in the phagocytic process. Yeast engulfment was completely inhibited by N-ethylmaleimide and iodoacetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2001

Identification and isolation of a full-length clone of mouse GMFB (Gmfb), a putative intracellular kinase regulator, differentially expressed in telencephalon.

We identified new transcribed sequences, using a differential display paradigm to select genes expressed in proliferating neuroblasts from mouse telencephalon at 10 days of embryonic development. In this systematic search, we isolated a 361-bp partial 3' untranslated region (3' UTR) homologous to the 3' UTR of the human gene encoding a putative intracellular kinase regulator, glia maturation factor beta (GMFB). We cloned a full-length, 4,311-bp mouse cDNA containing a 270-bp 5' UTR, a 3,615-bp 3' UTR, and an open reading frame of 426 nucleotides encoding a putative 142 amino-acid protein, identical to human GMFB, with the exception of two amino acids. This 4.3-kb transcript is present in a variety of adult tissues and is developmentally regulated as shown by Northern blot analysis. Differential expression in telencephalon was demonstrated by quantification of radioactive relative RT-PCR and confirmed by in situ hybridization. The isolation of this full-length clone of mouse Gmfb should facilitate investigation of the intracellular mechanisms involved in the development of telencephalon.

3' Untranslated Regions↗

PubMed2005

Non-invasive imaging of the endocrine pancreas (review).

The non-invasive imaging of the endocrine pancreas is currently considered by concerned institutions as a priority theme of research. Because the endocrine pancreas represents only about one percent of the pancreatic gland, highly specific tools are required for labelling the insulin-producing cells. The present review deals with the possible use of D-mannoheptulose for the non-invasive quantification of insulin-producing cells in the pancreas. This heptose is transported into hepatocytes and insulin-producing cells, but not other cell types, at the intervention of GLUT2. Its uptake coincides with the intracellular accumulation of acidic metabolites generated by phosphorylation of D-mannoheptulose. These metabolites remain in the islet cells after prolonged washing, suggesting that imaging of the pancreas at a relatively late time after intravenous administration of D-mannoheptulose could avoid significant extracellular contamination. Using tritiated D-mannoheptulose as tracer, a new method was designed for quantification of the total mass of insulin-producing cells in either isolated perfused rat pancreata or isolated pancreatic islets. Likewise, a preferential labelling of hepatocytes and insulin-producing transplanted cells can be achieved in vivo after administration of tritiated D-mannoheptulose. In the light of results obtained with 6-deoxy-6-iodo-D-glucose, it is proposed that 7-deoxy-7-iodo-D-mannoheptulose could be used for the non-invasive imaging of the endocrine pancreas.

Animals↗

PubMed2006

Separation and quantification of N-acetyl-l-cysteine and N-acetyl-cysteine-amide by HPLC with fluorescence detection.

N-acetyl-l-cysteine (NAC) is a well-known antioxidant that is capable of facilitating glutathione (GSH) biosynthesis and replenishing intracellular GSH under oxidatively challenging circumstances. N-acetyl-cysteine-amide (NACA), the amide form of NAC, is a newly designed and synthesized thiol-containing compound which is believed to be more lipophilic and permeable through cell membranes than NAC. The metabolic and antioxidant effects of these compounds in vitro and in vivo are under investigation. However, an analytical method that can separate and quantify both compounds simultaneously is not yet available, to the best of our knowledge. Because of their structural similarities, the two compounds are difficult to separate using earlier HPLC methods which were designed for NAC quantification. Therefore, the goal of this work was to develop an HPLC method with fluorescence detection for simultaneous quantification of NAC and NACA in biological blood and tissue samples. A gradient HPLC program with fluorescence detection (lambda(ex) = 330 nm, lambda(em) = 376 nm) using N-(1-pyrenyl)maleimide (NPM) as the derivatizing agent was developed. The calibration curves were linear over a concentration range of 25-5000 nm (r(2) > 0.997). The coefficients of variation for within-run precision and between-run precision ranged from 0.67 to 5.23% and for accuracy ranged from 0.98 to 10.54%; the percentage relative recovery ranged from 94.5 to 102.8%. This new method provides satisfactory separation of NAC and NACA, along with other biological thiols, in 20 min with a 5 nm limit of detection (LOD) per 5 microL injection volume.

Acetylcysteine↗

PubMedDate not supplied

Use of ATP bioluminescence to determine the bacterial sensitivity threshold to a bacteriocin.

A new ATP bioluminescence-based method was developed to determine the effectiveness of nisin on a sensitive strain of Lactococcus cremoris. The principle of the method is to quantify the release of adenylic-nucleotides (AN) by a sensitive strain under the action of the bacteriocin, with the complex luciferin-luciferase. Nisin-induced leakage of AN included ATP from a sensitive L. cremoris to the external medium immediately after the contact with the bacteria. The growth of L. cremoris was correlated with the extracellular AN content. The extracellular ATP and AN concentration exhibited a linear correlation to the logarithm of the nisin concentration. For the determination of the effectiveness threshold, the concentration of AN was more sensitive and more reliable than the direct quantification of ATP. The effectiveness threshold, corresponding to a 100% inhibition of L. cremoris growth, was obtained for a null concentration of intracellular nucleotides, i.e. for a AN(tot):AN(ext) ratio = 1. For an initial concentration of 1.4 x 10(7) bacteria/mL, the nisin effectiveness threshold is 3.4 +/- 0.01 mg nisin/L. It is possible to detect effectiveness threshold concentration by taking into account the physiological state of the cells.

Adenine Nucleotides↗

PubMed2002

Mutations in the UL97 ORF of ganciclovir-resistant clinical cytomegalovirus isolates differentially affect GCV phosphorylation as determined in a recombinant vaccinia virus system.

Mutations in the human cytomegalovirus (HCMV) UL97 phosphotransferase have been associated with ganciclovir (GCV) resistance due to an impairment of GCV monophosphorylation. Vaccinia virus recombinants (rVV) were generated that encoded different HCMV UL97 proteins (pUL97) with mutations previously detected in resistant HCMV clinical isolates at codons 460, 520, 592, 594, 595, 598 and 607. These rVVs allowed quantification of GCV phosphorylation catalyzed by the different mutated pUL97s. When compared to rVV-UL97 wild type, mean levels of residual intracellular GCV phosphorylation differed by a factor of 10 for the mutated UL97 proteins ranging from 5.2 to 51.8%. Mutations M460V (located in a UL97 region homologous to domain VIb of protein kinases) and H520Q (located in a cytomegalovirus-specific, functionally critical domain) were responsible for the lowest levels of residual GCV phosphorylation (9.3 and 5.2%). Mutations in a region homologous to the domain IX had a lower impact on GCV phosphorylation (15.8-51.8%). The relevance of pUL97 mutation G598S in inducing GCV resistance was demonstrated for the first time.

Animals↗

PubMed2006

Investigation of the origin of extracellular RNA in human cell culture.

We have used the human ECV 304 cell line to study the origin and fate of extracellular RNA (exRNA) in cell culture. Quantification of different extracellular RNA species using reverse transcription followed by quantitative PCR revealed a prevalent fraction of ribosomal RNAs. Comparison of intracellular and extracellular ribosomal RNA copy numbers allowed the calculation of the number of destroyed cells that would result in the corresponding number of extracellular rRNAs. Interestingly, this number was comparable to the amount of destroyed cells as determined by the measurement of extracellular lactate dehydrogenase activity.

Cell Line, Tumor↗

PubMed2000

Quantification of SNARE protein levels in 3T3-L1 adipocytes: implications for insulin-stimulated glucose transport.

Insulin-stimulates glucose transport in peripheral tissues by stimulating the movement ('translocation') of a pool of intracellular vesicles containing the glucose transporter Glut4 to the cell surface. The fusion of these vesicles with the plasma membrane results in a large increase in the numbers of Glut4 molecules at the cell surface and a concomitant enhancement of glucose uptake. It is well established that proteins of the VAMP- (synaptobrevin) and syntaxin-families play a fundamental role in the insulin-stimulated fusion of Glut4-containing vesicles with the plasma membrane. Studies have identified key roles for vesicle associated membrane protein-2 (VAMP2) and syntaxin-4 in this event, and more recently have also implicated SNAP-23 and Munc18c in this process. In this study, we have quantified the absolute levels of expression of these proteins in murine 3T3-L1 adipocytes, with the objective of determining the stoichiometry of these proteins both relative to each other and also in comparison with previous estimates of Glut4 levels within these cells. To achieve this, we performed quantitative immunoblot analysis of these proteins in 3T3-L1 membranes compared to known amounts of purified recombinant proteins. Such analyses suggest that in 3T3-L1 adipocytes there are approximately 374,000 copies of syntaxin 4, 1.15 x 10(6) copies of SNAP23, 495,000 copies of VAMP2, 4.3 x 10(6) copies of cellubrevin and 452,000 copies of Munc18c per cell, compared to previous estimates of 280,000 copies of Glut4. Thus, the main SNARE proteins involved in insulin-stimulated Glut4 exocytosis (syntaxin 4 and VAMP2) are expressed in approximately equimolar amounts in adipocytes, whereas by contrast the endosomal v-SNARE cellubrevin is present at approximately 10-fold higher levels and the t-SNARE SNAP-23 is also present in an approximately 3-fold molar excess. The implications of this quantification for the mechanism of insulin-stimulated Glut4 translocation are discussed.

3T3 Cells↗

PubMed2004

Dendritic oligoguanidines as intracellular translocators.

A series of polyguanidylated dendritic structures that can be used as molecular translocators have been designed and synthesized based on nonpeptide units. The dendritic oligoguanidines conjugated with fluorescein or with a green fluorescent protein (GFP) mutant as cargos were isolated and characterized. Quantification and time-course analyses of the cellular uptake of the conjugates using HeLa S3 and human cervical carcinoma cells reveal that the polyguanidylated dendrimers have comparable translocation efficiency to the Tat(49-57) peptide. Furthermore, the deconvolution microscopy image analysis shows that they are located inside the cells. These results clearly show that nonlinear, branched dendritic oligoguanidines are capable of translocation through the cell membrane. This work also demonstrates the potential of these nonpeptidic dendritic oligoguanidines as carriers for intracellular delivery of small molecule drugs, bioactive peptides, and proteins.

Biopolymers↗

PubMed2001

Quantification and sequencing of somatic deleted mtDNA in single cells: evidence for partially duplicated mtDNA in aged human tissues.

Single-cell PCR of the whole mitochondrial genome provides detailed information about intracellular clonal expansions of deleted mitochondrial DNA (DeltamtDNA), which contribute to aging of the muscle and possibly other tissues. Analysis of approximately 1400 cells from heart, diaphragm and skeletal muscle from 20 individuals without mitochondrial disease revealed that up to 25% of cells in a tissue sample may bear clonally expanded DeltamtDNA. Sequence analysis of >50 clonal DeltamtDNA reveals that about half of them lack the light strand origin of replication. This observation is puzzling since these molecules must have retained the ability to replicate in order to be able to undergo clonal expansion. We present evidence that such DeltamtDNA molecules may in fact exist in the cell as partially duplicated mtDNA (pdmtDNA) previously described in certain mtDNA disorders. In contrast to the 'originless' DeltamtDNA, the corresponding pdmtDNA do possess a light strand origin required for their propagation. Most pdmtDNA also possess an extra heavy strand origin, which may result in higher replication rate and thus provide a mechanism for expansion. Importantly, pdmtDNA are indistinguishable from DeltamtDNA in PCR assays routinely used to detect somatic mtDNA deletions in tissues of normally aged individuals. These results indicate that a substantial proportion of age-related mtDNA deletions reported in the literature may exist as or be derived from pdmtDNA.

Adolescent↗

PubMed1985

Intracellular type A retrovirus movement associated with an intact microtubule system.

Intracytoplasmic type A particles known to be precursors to type B retroviruses in murine, hamster and marsupial cells are closely associated with microtubules and microtubule organizing centres. In this publication, the active participation of microtubules in the intracellular transport of the particles to the cell surface has been examined in NIH 3T3 cells infected with M432 virus using vincristine sulphate (VCR) as inhibitor of microtubule polymerization. The release of virus at different times after exposure to VCR was quantified by reverse transcriptase determinations of cell supernatants and by electron microscopic quantification of the number of virions at the cell surface using freeze-dried whole cell replicas. These studies indicate that VCR inhibits both microtubule polymerization and virus release, and thus suggest that intact cytoplasmic microtubules are necessary for intracellular transport and release of virus.

Animals↗