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Results for “Intracellular quantification”

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PubMed2003

On-line monitoring of Phaffia rhodozyma fed-batch process with in situ dispersive Raman spectroscopy.

Since the yeast Phaffia rhodozyma was first described some 35 years ago, there has been significant interest in the development of commercial processes to exploit its ability to produce carotenoids (approximately 80% astaxanthin). However, the optimal conditions for carotenoid production are not well understood. A key limitation has been the lack of an appropriate sensor for on-line carotenoid quantification. In this study, an in situ Raman spectroscopy probe was used to monitor intracellular carotenoid production for three consecutive P. rhodozyma fed-batch experiments. Raman spectroscopy is particularly well suited to the study of carotenoids due to a resonance effect, which greatly enhances the intensity of the three fundamental carotenoid bands, nu(1) (1513 cm(-1), C(-) (-)C stretch), nu(2) (1154 cm(-1), C-C stretch), and nu(3) (1003 cm(-1), CH(3) rock). For all three cultures, the peak height of these bands was linearly correlated with intracellular carotenoid content (1 to 45 mg/L) to a precision of better than 5%, and the correlation from one experiment was directly applicable to others.

Algorithms↗

PubMed1996

Use of monoclonal antibodies to study interleukin-1 beta-converting enzyme expression: only precursor forms are detected in interleukin-1 beta-secreting cells.

We have generated a series of monoclonal antibodies (mAb) using recombinant interleukin (IL)-1 beta-converting enzyme (ICE) p20 and p10 subunits as immunogens. The mAb have been selected for further study based on their reactivity with ICE in transfected COS cells and their lack of cross-reactivity with TX, the closest ICE homolog known to date. Two anti-p20 and one anti-p10 mAb have been used to study ICE expression by Western blotting and immunodetection. In ICE-transfected COS cells, the mAb recognize the p45 ICE precursor and the maturation products (p20 or p10 subunits) for which they are specific. In monocytes and cell lines expressing ICE, only precursor forms are detected and intracellular immunostaining followed by confocal microscopy shows that they are located in the cytoplasm. Quantification experiments show that THP1 cells express approximately 67,000 molecules of ICE precursor per cell, with an estimated precursor to mature ratio of at least 100. In these cells as well as in monocytes, lipopolysaccharide stimulation did not change the pattern of ICE expression, although efficient secretion of mature IL-1 beta was measured. However, upon cell disruption, precursor maturation was observed. Our results, therefore, show that ICE is present in cells as a large pool of intracytoplasmic precursor, and that very limited amounts of mature ICE protein are present, but nevertheless sufficient to allow efficient IL-1 beta cleavage. Altogether, these observations suggest that post-translational maturation of the precursor protein could represent a specific step in the regulation of ICE enzymatic activity.

Amino Acid Sequence↗

PubMed1999

Tracers to investigate protein and amino acid metabolism in human subjects.

Three tracer methods have been used to measure protein synthesis, protein breakdown and protein oxidation at whole-body level. The method using L-[1-(13)C]leucine is considered the method of reference. These methods have contributed greatly to the existing knowledge on whole-body protein turnover and its regulation by feeding, fasting, hormones and disease. How exercise and ingestion of mixed protein-containing meals affect whole-body protein metabolism is still open to debate, as there are discrepancies in results obtained with different tracers. The contribution of whole-body methods to the future gain of knowledge is expected to be limited due to the fact that most physiological disturbances have been investigated extensively, and due to the lack of information on the relative contribution of various tissues and proteins to whole-body changes. Tracer amino acid-incorporation methods are most suited to investigate these latter aspects of protein metabolism. These methods have shown that some tissues (liver and gut) have much higher turnover rates and deposit much more protein than others (muscle). Massive differences also exist between the fractional synthesis rates of individual proteins. The incorporation methods have been properly validated, although minor disagreements remain on the identity of the true precursor pool (the enrichment of which should be used in the calculations). Arterio-venous organ balance studies have shown that little protein is deposited in skeletal muscle following a protein-containing meal, while much more protein is deposited in liver and gut. The amount deposited in the feeding period in each of these tissues is released again during overnight fasting. The addition of tracers to organ balance studies allows the simultaneous estimation of protein synthesis and protein breakdown, and provides information on whether changes in net protein balance are caused primarily by a change in protein synthesis or in protein breakdown. In the case of a small arterio-venous difference in a tissue with a high blood flow, estimates of protein synthesis and breakdown become very uncertain, limiting the value of using the tracer. An additional measurement of the intracellular free amino acid pool enrichment allows a correction for amino acid recycling and quantification of the inward and outward transmembrane transport. However, in order to obtain reliable estimates of the intramuscular amino acid enrichment and, therefore, of muscle protein synthesis and breakdown in this so-called three-pool model, the muscle should be freeze-dried and the resulting fibres should be freed from connective tissue and small blood clots under a dissection microscope. Even when optimal precautions are taken, the calculations in these tracer balance methods use multiple variables and, therefore, are bound to lead to more variability in estimates of protein synthesis than the tracer amino acid incorporation methods. In the future, most studies should focus on the measurement of protein synthesis and breakdown in specific proteins in order to understand the mechanisms behind tissue adaptation in response to various stimuli (feeding, fasting, exercise, trauma, sepsis, disuse and disease). The tracer laboratories, therefore, should improve the methodology to allow the measurement of low tracer amino acid enrichments in small amounts of protein.

Amino Acids↗

PubMed2003

Motility analysis of pancreatic adenocarcinoma cells reveals a role for the atypical zeta isoform of protein kinase C in cancer cell movement.

The acquisition of an invasive and metastatic phenotype is accompanied by profound alterations of intracellular mechanisms controlling cell movement. Analysis of quantitative parameters of cell motility in cancer cells may help in the identification of intracellular signaling events determining invasion and metastasis. Here we developed a novel procedure of quantification of cell motility based on time-lapse video microscopy and digital image analysis. Three kinetic parameters, including area change, plasma membrane remodeling, and speed of linear movement, are quantified and combined in one single, time-normalized value we defined motility score (MS). Through calculation of the MS for various human pancreatic adenocarcinoma cell subclones, we identified clones characterized by low or high spontaneous motility in vitro. Analysis of the signaling mechanisms involved in the regulation of pancreatic adenocarcinoma cell motility showed that the atypical zeta isozyme of the serine-threonine protein kinase C (PKC) plays a critical role in maintaining a high MS in motile subclones, as demonstrated by the inhibitory effect of cell permeable peptides with sequence corresponding to the pseudosubstrate inhibitory region of the atypical zeta PKC. Other PKC isozymes, either classic or novel, seem not involved. Furthermore, biochemical analysis showed that in motile cells, zeta PKC is constitutively associated with the plasma membrane, whereas in nonmotile cells, zeta PKC is totally excluded from the plasma membrane. These data suggest that the disregulation of the function of atypical zeta PKC might be involved in the acquisition of an invasive and metastatic phenotype in pancreatic adenocarcinoma cells.

3T3 Cells↗

PubMed2001

Transcript quantification based on chemical labeling of RNA associated with fluorescent detection.

A general method for RNA measurement, based on chemical labeling of RNA with digoxigenin (without retrotranscription), has been established. Labeled RNA is hybridized with nylon membranes containing spot blots of PCR-amplified gene fragments and the fluorescence detection is mediated via specific anti-digoxigenin antibody coupled to alkaline phosphatase. The method was optimized in order to be quantitative, and high precision (less than 24% error) was obtained, allowing analysis of relatively small changes in gene expression. When the quantity of cellular RNA used in this method is maintained constant and the amount of RNA in the cell determined, the true intracellular transcript concentrations can be determined, rather than simple abundance of a messenger in RNA population. This RNA quantification technique was extended to macroarrays blotted automatically and the validity of the method was tested by comparison with expression data obtained by Northern blotting.

Blotting, Northern↗

PubMed1992

Quantification and localization of ornithine decarboxylase in the embryonic palate.

Ornithine decarboxylase (ODC; EC4.1.1.17), the key enzyme in polyamine biosynthesis, and intracellular polyamines increase rapidly and markedly in tissues and cells that are actively proliferating as well as differentiating and decrease as these processes cease. ODC activity has also been implicated as playing a role in the proliferation and differentiation of cells derived from the developing palate. Ornithine decarboxylase activity was thus quantified and ODC localized in the developing murine palate in vivo. Levels of ODC activity showed little variation during the ontogeny of the palate, averaging 126 pmol CO2/mg protein/hr. When difluoromethylornithine (DFMO), an irreversible inhibitor of ODC activity, was administered to pregnant mice throughout the period of palate development (days 11-14), palatal tissue ODC activity was reduced by 85%. No craniofacial malformations were observed, however. The lack of a teratogenic effect by DFMO treatment could be due to sufficient remaining ODC activity in craniofacial tissue and/or maintenance of intracellular polyamine levels by the activity of a polyamine transport system. The activity of this system was demonstrated by the ability of palatal tissue in vivo to take up radiolabeled putrescine. The presence of a polyamine transport system was previously suggested by the demonstration of such a system in palate mesenchymal cells in vitro. Dramatic temporal and spatial shifts in tissue patterns of immunolocalization for ODC in developing palatal tissue were also seen. Immunostaining for ODC was evenly distributed in oral, nasal, and medial edge palate epithelial cells on day 12 of gestation. The basal aspects of epithelial cells were, however, more intensely stained. Mesenchymal cells exhibited a peri-nuclear immunostaining pattern. On days 12 and 13 of gestation, the staining patterns for ODC in palate epithelial and mesenchymal cells were comparable. On day 14 of gestation, all regions of the palate epithelium, particularly the medial edge epithelia, were immunostained for ODC, whereas the intensity of staining in the mesenchymal cells was significantly reduced. This study represents essential initial observations toward understanding the role that ODC may play in normal craniofacial development.

Animals↗

PubMed2002

Nitric oxide-mediated chondrocyte cell death requires the generation of additional reactive oxygen species.

OBJECTIVE: Chondrocyte cell death, possibly related to increased production of endogenous nitric oxide (NO), has been observed during the pathogenesis of osteoarthritis and rheumatoid arthritis. The purpose of this study was to investigate the potential role of NO in causing chondrocyte cell death and to determine the contribution of other reactive oxygen species (ROS). METHODS: Cell death and cytotoxicity were evaluated in human articular chondrocytes in response to various NO donor compounds with and without agents that stimulate or inhibit the production of additional ROS using both the alginate bead and the monolayer culture systems. Cell death was quantified by a total cell count with fluorescent labels, and cytotoxicity was measured as a function of cellular NADH- and NADPH-dependent dehydrogenase activity. To determine if the redox status of the chondrocyte could influence the observed effect of NO, cells were preincubated for 24 hours in L-cystine- and glutathione (GSH)-depleted media to reduce intracellular GSH levels, a major defense mechanism against oxidative stress. Apoptosis was analyzed with the quantification of histone-associated DNA fragments. RESULTS: Treatment of chondrocytes with peroxynitrite (ONOO-), 3-morpholinosydnonimine (SIN-1), and sodium nitroprusside (SNP) resulted in apoptotic cell death at concentrations of 0.5 mM, 1.0 mM, and 0.5 mM, respectively. In contrast, treatment of chondrocytes with diazeniumdiolates (or the "NOC" compounds, NOC-5 and NOC-12) at concentrations as high as 2.0 mM did not cause cell death. Furthermore, NOC-5 and NOC-12, at all concentrations tested (0.125-2.0 mM), could prevent cell death caused by oxidative stress. Selective ROS scavengers protected against cell death caused by either SIN-1 or ONOO-; however, no protection could be afforded against the cytotoxicity of SNP with any of the ROS scavengers tested. CONCLUSION: These results show that NO by itself is not cytotoxic to cultured chondrocytes and can even be protective under certain conditions of oxidative stress. Chondrocyte cell death from NO occurs under conditions where other ROS are also generated.

Alginates↗

PubMed1996

Isolation of heavy endosomes from dog proximal tubules in suspension.

During the preparation of a suspension of dog kidney proximal tubules by collagenase treatment, an uptake of FITC-albumin was demonstrated. This process is attributed to the activation of receptor-mediated endocytosis leading to the appearance of FITC-albumin into intracellular vesicular structures. The isolation of brush border membrane vesicles (BBMV) from the dog kidney proximal tubules in suspension by the magnesium precipitation technique leads to the copurification of a large population of endosomes. These endosomes were separated from BBM vesicles by a technique involving wheat-germ agglutinin. The enrichment in BBM markers and in bafilomycin-sensitive ATPase activity was comparable in endosomes and BBM vesicles. However, the acridine orange acidification assay showed a V-type ATPase-dependent acidification in endosomes but not in BBMV, demonstrating a different orientation of the proton pumps in these structures. SDS-PAGE analysis also showed significant differences in protein pattern of vesicles and endosomes. The most notable difference was the presence of 42-44 kDa and 20-24 kDa proteins in BBMV and their complete absence in endosomes. Western blot analysis identified these proteins as actin and RhoA, among other small proteins, respectively. Western blot experiments also demonstrated a different distribution of beta-COP, beta-adaptin, and RhoGDI in vesicles and endosomes. The morphological aspect (electron microscopy) and sedimentation of endosomes in a 50% Percoll gradient identified these structures as "heavy endosomes" (buoyant density D = 1.036 g/ml). Flow cytometry analysis of heavy endosomes purified from tubules isolated in presence of FITC-albumin showed the presence of FITC-albumin in up to 92% of these intracellular organelles. Western blot analysis using anti-FITC and anti-collagenase antibodies allowed quantification of the FITC-albumin and collagenase A in the purified endosomes. Our results indicate that heavy endosomes are formed during the preparation of the proximal tubules following activation of receptor-mediated endocytosis, probably by soluble proteins. The suspension of tubules thus offers a experimental tool to study the protein reabsorption and traffic of endosomal vesicles in the proximal tubules.

Animals↗

PubMed2003

In vitro effects of ascorbate and Trolox on the biocompatibility of dental restorative materials.

Previous in vitro studies on the cytotoxicity of eight dental restorative materials including composites, compomers, resin-modified glass ionomer cements and glass ionomer cements have demonstrated a depletion of intracellular glutathione in gingival fibroblasts incubated with eluates of these materials and a protective effect of N-acetylcysteine. In the present study, we investigate the effects of two other antioxidants: ascorbate and Trolox. It was found that Trolox reduced the cytotoxicity induced by resin-based biomaterial eluates. In contrast, ascorbate increased in a dose-dependent manner the toxic effect of all eluates except for Z100 MP and Tetric flow (composites). The effect of D-mannitol was studied for GC FUJI II and was found to neutralize the additional toxic effect of ascorbate. Ascorbate increased the depletion of intracellular glutathione of these dental material eluates (between 17% and 24%, depending on the material). Quantification of metal ions in the dental material eluates showed the presence of significant amounts of aluminum and iron in GC FUJI II > photac fil > GC FUJI II LC > F2000. The mechanism of this increased cytotoxicity could be explained by the Fenton reaction resulting from the pro-oxidant effect of ascorbate in the presence of iron (transition metal ions) and/or aluminum.

Aluminum↗

PubMed2003

Profiling receptor tyrosine kinase activation by using Ab microarrays.

Signal transduction in mammalian cells is mediated by complex networks of interacting proteins. Understanding these networks at a circuit level requires devices to measure the amounts and activities of multiple proteins in a rapid and accurate manner. Ab microarrays have previously been applied to the quantification of labeled recombinant proteins and proteins in serum. The development of methods to analyze intracellular signaling molecules on microarrays would make Ab arrays widely useful in systems biology. Here we describe the fabrication of multiplex Ab arrays sensitive to the amounts and modification states of signal transduction proteins in crude cell lysates and the integration of these arrays with 96-well microtiter plate technology to create microarrays in microplates. We apply the Ab arrays to monitoring the activation, uptake, and signaling of ErbB receptor tyrosine kinases in human tumor cell lines. Data obtained from multicolor ratiometric microarrays correlate well with data obtained by using traditional approaches, but the arrays are faster and simpler to use. The integration of microplate and microarray methods for crude cell lysates should make it possible to identify and analyze small molecule inhibitors of signal transduction processes with unprecedented speed and precision. We demonstrate the future potential of this approach by characterizing the action of the epidermal growth factor receptor inhibitor PD153035 on cells by using Ab arrays; direct scale-up to array-based screening in 96- and 384-well plates should allow small molecules to be identified with specific inhibitory profiles against a signaling network.

Animals↗

PubMed1989

Changes in concentrations of coenzyme F420 analogs during batch growth of Methanosarcina barkeri and Methanosarcina mazei.

Coenzyme F420 has been assayed by high-performance liquid chromatography with fluorimetric detection; this permits quantification of individual coenzyme F420 analogs whilst avoiding the inclusion of interfering material. The total intracellular coenzyme F420 content of Methanosarcina barkeri MS cultivated on methanol and on H2-CO2 and of Methanosarcina mazei S-6 cultured on methanol remained relatively constant during batch growth. The most abundant analogs in M. barkeri were coenzymes F420-2 and F420-4, whilst in M. mazei coenzymes F420-2 and F420-3 predominated. Significant changes in the relative proportions of the coenzyme F420 analogs were noted during batch growth, with coenzymes F420-2 and F420-4 showing opposite responses to each other and the same being also true for coenzymes F420-3 and F420-5. This suggests that an enzyme responsible for transferring pairs of glutamic acid residues may be active. The degradation fragment FO was also detected in cells in late exponential and stationary phase. Coenzyme F420 analogs were present in the culture supernatant of both methanogens, in similar proportions to that in the cells, except for FO which was principally located in the supernatant.

Chromatography, High Pressure Liquid↗

PubMed1998

Accumulation of amphotericin B in human macrophages enhances activity against Aspergillus fumigatus conidia: quantification of conidial kill at the single-cell level.

A cytofluorometric assay that allowed assessment of damage to phagocytosed Aspergillus fumigatus conidia at the single-cell level was developed. After ingestion by monocyte-derived macrophages (MDMs), conidia were reisolated by treatment of the cells with streptolysin O, a pore-forming toxin with lytic properties on mammalian cells but not on fungi. The counts obtained by staining of damaged conidia with propidium iodide and quantification by cytofluorometry correlated with colony counts. By the use of this method, we demonstrate that MDMs differentiated in vitro by low-dose granulocyte-macrophage colony-stimulating factor and gamma interferon have only a limited capacity to damage Aspergillus conidia in vitro. The killing rate 12 h after phagocytosis was found to be only 10 to 15%. However, intracellular loading of the phagocytes with amphotericin B (AmB) dose dependently enhanced the anticonidial activity. Preincubation of macrophages with only 1 microg of AmB per ml resulted in an uptake of 18 fg of AmB/cell, leading to killing rates of 50 to 60%. The experimental protocol provides a new tool for the rapid quantification of anticonidial activity against A. fumigatus in vitro. Intracellular accumulation of AmB may represent an important factor underlying the efficacy of this antifungal drug in the prophylaxis and treatment of Aspergillus infections.

Amphotericin B↗

PubMed1998

A cytofluorometric method for the simultaneous detection of both intracellular and surface antigens of apoptotic peripheral lymphocytes.

The aim of this study was to define a simple and reliable method to detect simultaneously surface and intracellular antigens in apoptotic peripheral human lymphocytes. This approach requires a permeabilizing procedure for intracellular access of mAbs, which raises the important question of the influence of this procedure on parameters which identify apoptotic cells and on the surface expression of antigens. We compared the effects of three currently used permeabilizing methods (saponin quillaia bark 0.05%, Triton X-100 0.1, ethanol 70%) on the quantification of apoptotic lymphocytes, defined according to FSC/SSC criteria or following 7-AAD staining, and on the detection of surface CD3, CD4, CD8, Fas, CD45R0 molecules. The combined detection of these surface antigens with intracellular molecules, including Bcl-2 and cytokines (IFNgamma, TNFalpha, IL-2) was also analysed in the context of these three permeabilizing procedures. All the experiments were performed on PBMC from HIV-infected donors, known to undergo excessive apoptosis following short-term culture. We report that permeabilization with saponin is the only procedure which allows: (1) the preservation of lymphocyte morphology determined by the FSC/SSC parameters; (2) the quantification of apoptotic lymphocytes following 7-AAD staining; (3) a reliable surface immunophenotyping, maintaining a good antibody binding capacity (ABC); (4) the proper detection of intracellular membrane bound antigens (Bcl-2) and intracellular cytokines (IFNgamma, TNFalpha, IL-2); (5) the combined detection of apoptotic nuclei, surface antigens and intracellular molecules. Altogether these observations demonstrate that the simultaneous analysis of extracellular and intracellular antigens in apoptotic cells belonging to a complex lymphoid populations such as PBMC can be readily overcome provided the detergent used for cell permeabilization is appropriate and the successive staining procedures performed in a defined order.

Antibodies, Monoclonal↗

PubMedDate not supplied

Non-viral vector-mediated uptake, distribution, and stability of chimeraplasts in human airway epithelial cells.

BACKGROUND: Chimeraplasty is a novel methodology that uses chimeric RNA/DNA oligonucleotides (chimeraplasts) to stimulate genomic DNA repair. Efficient uptake and nuclear localization of intact chimeraplasts are key parameters to achieve optimal correction of mutation defects into specific cell types. METHODS: A 5'-end FITC-labeled 68-mer RNA/DNA oligonucleotide was complexed with the polycation polyethylenimine (PEI) and the cationic lipids Cytofectin and GenePorter. Flow cytometry was employed to evaluate chimeraplast uptake under different conditions. Intracellular chimeraplast distribution and co-localization with endocytosis markers were assessed by confocal microscopy. Relative quantification of chimeraplast metabolism was performed by denaturing PAGE and GeneScan(trade mark) analysis. RESULTS: In airway epithelial cells, optimized chimeraplast uptake reached near 100% efficiency with the carriers tested. However, chimeraplast nuclear localization could only be achieved using PEI or Cytofectin. Chimeraplast/GenePorter lipoplexes were retained in the cytoplasm. PEI polyplexes and Cytofectin lipoplexes displayed different uptake rates and internalization mechanisms. Chimeraplast/PEI polyplexes were internalized at least partially by fluid-phase endocytosis. In contrast, phagocytosis may have contributed to the internalization process of large-sized chimeraplast/Cytofectin lipoplexes. Moreover, significant chimeraplast degradation was detected 24 h after transfection with both PEI polyplexes and Cytofectin lipoplexes, although the latter seemed to confer a higher degree of protection against nuclease degradation. CONCLUSION: Both Cytofectin and PEI are efficient for chimeraplast nuclear uptake into airway epithelial cells. However, despite the distinct structures and trafficking pathways of the corresponding complexes, none of them could prevent nuclease-mediated metabolism of the chimeric oligonucleotides. These findings should be taken into account for future investigations of chimeraplast-mediated gene repair in airway epithelial cells.

Base Sequence↗

PubMed2002

Detection of dengue virus replication in peripheral blood mononuclear cells from dengue virus type 2-infected patients by a reverse transcription-real-time PCR assay.

While dengue virus is thought to replicate in mononuclear phagocytic cells in vivo, attempts to detect it in peripheral blood mononuclear cells (PBMC) by virus isolation or antigen detection have had variable and generally low rates. In this study, we developed a reverse transcription (RT)-real-time PCR assay to quantify positive- and negative-sense RNA of dengue virus type 2 within the cells. The assay includes an RT step using either sense or antisense primer followed by a real-time PCR step using the designed primers and probe, which target a capsid region highly conserved in dengue virus type 2 strains. It can be used to monitor the dynamic change of intracellular dengue virus RNA species during the course of infection. When this assay is employed in quantification of dengue virus RNA species in PBMC from 10 patients infected with dengue virus type 2, both positive- and negative-sense dengue RNA can be detected, indicating that dengue virus is actively replicating in PBMC in vivo. Moreover, the amounts of negative-sense dengue virus RNA in PBMC correlate very well with the viral load of dengue virus in plasma, suggesting that quantification of negative-sense dengue virus RNA in PBMC may provide another indicator of dengue virus replication in vivo. Use of this convenient, sensitive, and accurate method of quantification in clinical samples from patients with different disease severity would further our understanding of the pathogenesis of dengue.

Animals↗

PubMed2002

Anticoagulants and other preanalytical factors interfere in plasma nitrate/nitrite quantification by the Griess method.

Nitric oxide (NO) is a signal molecule with functions such as neurotransmission, local vascular relaxation, and anti-inflammation in many physiological and pathological processes. Various factors regulate its intracellular lifetime. Due to its high reactivity in biological systems, it is transformed in the bloodstream into nitrates (NO(-)(3)) by oxyhemoglobin. The Griess reaction is a technically simple method (spectrophotometric, 540 nm) for the analysis of nitrites (NO(-)(2)) in aqueous solutions. We studied the interference of common anticoagulants in the quantification of nitrate and nitrite in plasma samples by the Griess method. We obtained rat plasma using heparin or sodium EDTA as anticoagulants, then added, or otherwise, known NO(-)(3) amounts in order to calculate their recovery. We also studied the effect of ultra-filtration performed before Griess reaction on plasma and aqueous solutions of various anticoagulants (heparin, EDTA, and also sodium citrate) to compare the recoveries of added NO(-)(3) or NO(-)(2). We used standards of NO(-)(3) or NO(-)(2) for quantification. We conclude that: (i) The bacterial nitrate reductase used to reduce NO(-)(3) to NO(-)(2) is unstable in certain storage conditions and interferes with different volumes of plasma used. (ii) The ultrafiltration (which is sometimes performed before the Griess reaction) of plasma obtained with EDTA or citrate is not recommended because it leads to overestimation of NO(minus sign)(3). In contrast, ultrafiltration is necessary when heparin is used. (iii) The absorbance at 540 nm attributed to plasma itself (basal value or background) interferes in final quantification, especially when ultrafiltration is not performed. For the quantification of plasma NO(-)(3) we recommend: sodium EDTA as anticoagulant, no ultrafiltration of plasma, and measurement of the absorbance background of each sample.

Animals↗

PubMed1983

The large intracellular pool of asialoglycoprotein receptors functions during the endocytosis of asialoglycoproteins by isolated rat hepatocytes.

The function of intracellular asialoglycoprotein receptors during the endocytosis of asialo-orosomucoid in isolated hepatocytes was assessed by following changes in the occupancy of intracellular receptors. Unoccupied total cellular (inside and surface) or surface receptors were quantified at 0 degrees C by the binding of 125I-asialo-orosomucoid in the presence or absence, respectively, of digitonin. Freshly isolated cells had about 17% of their total receptors on the surface. After incubation at 37 degrees C, the receptor distribution changed to 25 to 50% on the cell surface and 50 to 75% inside the cell. At 37 degrees C, the average total number of receptors/cell was 4.5 x 10(5). Dissociation constants, determined from equilibrium binding studies in the presence or absence of digitonin to assess total or surface receptors, were identical (5.4 +/- 1.4 and 5.6 +/- 1.1 x 10(-9) M, respectively). In the presence of asialo-orosomucoid at 37 degrees C, there was both a time- and a concentration-dependent decrease in surface and intracellular receptor activity. This receptor activity decrease was reversed by removing asialo-orosomucoid from the medium or by washing the digitonin-permeabilized cells with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prior to quantification of receptor activity. Within 1 to 2 h in the presence of excess asialo-orosomucoid, a steady state was attained in which approximately 70% of the intracellular receptors were occupied. The kinetics of receptor activity recovery on the cell surface after internalization of a pulse of ligand is different than the rate of recovery of internal receptor activity. The results suggest that all of the internal asialoglycoprotein receptors are functional and participate during endocytosis. Internal receptors may be functionally equivalent to those on the surface or they may serve a reservoir or routing function for internalized ligand.

Animals↗

PubMed1994

Clinical and biochemical aspects of uric acid overproduction.

Purine nucleotides are synthesized and degraded through a regulated series of reactions which end in the formation of uric acid. Increased uric acid synthesis may be the result of two major pathophysiological disorders: increased de novo purine synthesis and enhanced purine nucleotide degradation, both of which may be the result of an increased or decreased enzyme activity. In addition, some conditions and disorders associated with uric acid overproduction have been recognized as the result of increased ATP degradation or decreased synthesis of ATP. The clinical manifestations of the diseases leading to excess uric acid synthesis are heterogenous, but symptoms related to uric acid overproduction are always secondary to the precipitation of crystals in soft tissues, joints, and the kidney excretory system. In clinical practice, serum urate concentration and urinary uric acid excretion are used to assess uric acid synthesis, taking into account that a purine-rich diet can be a confounding variable. Quantification of uric acid precursors, such as adenosine, inosine, guanosine, hypoxanthine, and xanthine, in biological fluids and intracellular nucleotides has provided further insight into the metabolic disturbances underlying disorders associated with uric acid overproduction. Additional studies are necessary to define precisely the metabolic derangement in idiopathic uric acid overproduction and to assess fully the consequences of increased purine nucleotide degradation, such as free-radical formation, increased adenosine synthesis, and reduced synthesis of signal transducers.

Adenosine Triphosphate↗