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PubMed2006

A novel function for selenium in biological system: selenite as a highly effective iron carrier for Chinese hamster ovary cell growth and monoclonal antibody production.

As the market for biopharmaceuticals especially monoclonal antibodies (MAbs) rapidly grows, their manufacturing methods are coming under increasing regulatory scrutiny, particularly due to concerns about the potential introduction of adventitious agents from animal-sourced components in the media used for their production in mammalian cell culture. Chinese hamster ovary (CHO) cells are by far the most commonly used production vehicles for these recombinant glycoproteins. In developing animal-component free media for CHO and other mammalian cell lines, the iron-transporter function of serum or human/bovine transferrin is usually replaced by certain organic or inorganic chelators capable of delivering iron for cell respiration and metabolism, but few of them are sufficiently effective. Selenium is a well-known essential trace element (TE) for cell growth and development, and its positive role in biological system includes detoxification of free radicals by activating glutathione peroxidase. In cell culture, selenium in the form of selenite can help cells to detoxify the medium thus protect them from oxidative damage. In this presentation, we describe the discovery and application of a novel function of selenite, that is, as a highly effective carrier to deliver iron for cell growth and function. In our in-house-developed animal protein-free (APF) medium for CHO cells, using an iron-selenite compound to replace the well-established tropolone delivery system for iron led to comparable or better cell growth and antibody production. A high cell density of >10 x 10(6) viable cells/mL and excellent antibody titer of approximately 3 g/L were achieved in 14-day fed-batch cultures in shake flasks, followed by successful scale-up to stirred bioreactors. The preparation of the commercially unavailable iron-selenite compound from respective ions, and its effectiveness in cell-culture performance, were dependent on reaction time, substrates, and other conditions.

Animals↗

PubMedDate not supplied

Cytotoxic and DNA-inhibitory effects of iron chelators on human leukaemic cell lines.

Cytotoxic and DNA synthesis inhibitory effects of several iron chelators with different physicochemical properties have been tested in four myeloid leukaemic cell lines (U937, K562, ML2 and HL60). The small lipophilic chelators 8-hydroxyquinoline, tropolone and omadine at 2 X 10(-5) M, caused substantial inhibition of labelled leucine and thymidine uptake into cells and also cell death following 4-h incubation. These effects were approximately 10-fold increased when the drugs were pre-incubated with equimolar amounts of iron. Iron alone and hydrophilic chelators e.g. desferrioxamine had insignificant cytotoxic and DNA synthesis inhibitory effects under the same conditions.

Cell Division↗

PubMed2001

Fungicidal activity of beta-thujaplicin analogues.

The fungicidal activity of analogues of beta-thujaplicin, a natural product responsible for the durability of heartwood of several cupressaceous trees, was investigated in vitro on the growth of different white and brown rot fungi involved in wood biodegradation, Coriolus versicolor, Phanerochaete chrysosporium, Poria placenta and Gloephyllum trabeum. The study shows that 2-hydroxycyclohepta-2,4,6-trienone (tropolone), easily prepared according to a literature procedure, possesses interesting fungicidal activity when compared to beta-thujaplicin, azaconazole, tebuconazole and copper oxine, which suggests this compound should be examined further as a potential biocide for wood preservation.

Anti-Infective Agents↗

PubMed1998

Effect of siderophores, catecholamines, and catechol compounds on Listeria spp. Growth in iron-complexed medium.

Almost all bacteria require iron for growth and virulence expression. However, Listeria spp. do not produce any siderophore for iron acquisition. Representative strains of each of the six species of Listeria were examined for their ability to use various compounds as iron suppliers in iron-restricted medium. Here we show that L. monocytogenes, L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, and L. grayi were able to use exogenous siderophores and various catechol ligands, including catecholamines, to overcome growth inhibition induced by tropolone, an iron chelating agent. In contrast, no growth promoting effect was observed with normetanephrine or 4-hydroxy-3-methoxyphenylglycol-piperazine salt, which indicates that the o-diphenol function of the ligand must be free to allow iron acquisition. Furthermore, we demonstrate that catecholamines do not act through specific bacterial receptors, because no difference in growth stimulation was observed between [+]- and [-]-norepinephrine. These results show that utilization of a variety of catechol compounds to acquire iron is a general phenomenon in the genus Listeria.

Catecholamines↗

PubMed2000

Hinokitiol, a selective inhibitor of the platelet-type isozyme of arachidonate 12-lipoxygenase.

Hinokitiol (4-isopropyltropolone), a constituent of Japanese cypress, reversibly inhibited platelet-type 12-lipoxygenase with an IC(50) of 0.1 microM, and the enzyme activity was almost lost at 1 microM. The compound was much less active with other lipoxygenase enzymes with higher IC(50) values (leukocyte-type 12-lipoxygenase, 50 microM; soybean lipoxygenase, 17 microM; 15-lipoxygenase-1, >100 microM; 5-lipoxygenase, 17 microM). Hinokitiol up to 100 microM had almost no effect on cyclooxygenases-1 and -2. Their structure-activity relationship examined with various tropolone derivatives indicated the requirements of the 2-hydroxyl group and 4-alkyl group for the potent and selective inhibition of platelet-type 12-lipoxygenase.

Animals↗

PubMed1996

Homogeneous or heterogeneous distribution of systemically administered adrenaline: organ dependence.

In incubation experiments it was shown that exogenous adrenaline or noradrenaline does not distribute homogeneously into the adrenergic varicosities of the rat vas deferens (wall with thick and compact muscle layer) but does distribute homogeneously in the rat spleen capsule (thin and loose muscle layer, containing more extracellular space than the vas deferens). To circumvent any hypothetical role of the muscular layer in the distribution of the amine, 100 micrograms.kg-1.h-1 adrenaline was administered to rats in vivo either i.v. (during 90 min) or i.p. (under pentobarbital anaesthesia, an Alzet minipump was implanted which delivered that dose during 6 days). The rats also received 100 mg.kg-1 pargyline (to inhibit MAO) and 100 mg.kg-1 tropolone (to inhibit COMT). At the end of adrenaline administration, vasa deferentia and spleen capsule were removed, washed and then exposed to 100 mumol.l-1 tyramine for 20 min. At the end of this exposure, the ratio noradrenaline/adrenaline in the tissue and in the medium was compared. In the vas deferens both after i.v. and i.p. administration of adrenaline, the ratio noradrenaline/adrenaline was about 3 times higher in the medium than in the tissue, while in the spleen capsule the ratio noradrenaline/adrenaline was not significantly different in the medium and in the tissue. We conclude that, even when the amine reaches the storage sites from the blood, it distributes homogeneously in the spleen capsule and heterogeneously in the vas deferens, perhaps because there are more than one kind of storage vesicles in the vas deferens.

Adrenergic Agents↗

PubMed1994

Quantification of pulmonary uptake of indium-111 labelled granulocytes in inflammatory bowel disease.

This study describes a method for quantifying the pulmonary trapping of indium-111 labelled polymorphonuclear (PMN) cells in patients with inflammatory bowel disease (IBD) in comparison to non-inflamed controls. Twenty patients with extensive IBD were studied by 111In-PMN scintigraphy. Gamma-camera images were obtained at 2.5-4 h (early) and 20-25 h (late) after the injection of autologous PMNs labelled in plasma with 111In-tropolonate. Local uptake in the chest, iliac bone marrow, spleen and liver was quantified as the counts per pixel per second per MBq of injected 111In for both early and late scans. Fourteen subjects without inflammatory disease were studied as controls. IBD patients showed significantly greater loss of splenic activity between early and late scans compared with controls (mean +/- SD: -35.7% +/- 16.6% versus -4.5% +/- 6.1%, P < 0.001). There was no significant difference between control and IBD groups with respect to liver and bone marrow uptake on both early and late scans. Chest uptake was significantly higher in patients with IBD on both early (6.4 +/- 1.6 cps/MBq/pix) and late (5.6 +/- 1.5 cps/MBq/pix) scans, compared with the controls (4.8 +/- 1.3 cps/MBq/pix, P < 0.005 and 3.4 +/- 1.0 cps/MBq/pix, P < 0.001 respectively). The chest uptake in the control group on the late scans demonstrated a significant linear correlation with iliac uptake (y = 0.23x + 0.41, r = 0.87, n = 14). Assuming in controls that there is no parenchymal uptake of 111In, this regression enables an estimate to be made, based on iliac counts, of the count rate from bone marrow in the chest wall.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1985

Lymphocyte labelling with indium: cytotoxicity studies.

Viability studies on lymphocytes labelled with indium In111 using oxine as a ligand showed impairment as measured by trypan-blue assessment and rosetting ability. In addition, lymphocyte response to phytohaemagglutinin stimulation as measured by tritiated-thymidine uptake was also impaired at levels where adequate cell labelling had taken place. Cadmium toxicity was not noticed, and the use of tropolone as a ligand offered possibilities of reduced cellular toxicity. Such cytotoxicity may not have been important in earlier reported studies on granulocytes where the large numbers available for in vivo work and the short periods of study still allowed useful conclusions to be drawn. However, because of the prolonged lifespan of the human lymphocyte, the cytotoxic effects of the processing might well make the long-term studies which would be of interest much less reliable for clinical assessment.

Cytotoxicity, Immunologic↗

PubMed1988

Evaluation of 111In labelled white blood cells by in vitro functional tests and electron microscopy. Comparison of three labelling methods.

We have studied the influence of granulocyte labelling with commercially available 111In-oxine, tropolone (trop) or home made 111In-Mercapto pyridine (Merc) prepared by the method of Thakur (1985) on the cell structure by electron microscopy and on the cell function by enzymatic tests, random migration, chemotaxis, phagocytosis and bactericidal activity. The granulocytes were labelled with 400 microCi 111In-oxine in saline or 111In-trop or Merc in plasma. The effect of the chelating agents with and without addition of the tracer was studied (n = 4) with varying concentrations: 5-10 micrograms/ml oxine, 10-160 micrograms/ml trop and 1-4 micrograms/ml Merc. Chemotaxis and random migration were not affected by 111In-trop and clearly suppressed by 111In-oxine and Merc; the other tests were normal. The cell structure was disturbed by Merc. The labelling efficiency was excellent with oxine (90%), acceptable with trop (30%-80%) and poor with Merc (10%-25%). Without 111In, chemotaxis and random migration were normal up to a concentration of 80 micrograms/ml trop, 8.5 micrograms/ml oxine and 1 microgram/ml Merc. With addition of 111In, chemotaxis and random migration were unaffected up to 80 micrograms/ml by trop and markedly suppressed by Merc and oxine. It is concluded that labelling with 111In-trop assures intact cells.

Granulocytes↗

PubMed1995

Technetium-99m and indium-111 double labelling of granulocytes for kinetic and clinical studies.

A new technique of labelling granulocytes with both technetium-99m hexamethylpropylene amine oxime (HMPAO) and indium-111 in a single protocol was developed in order to exploit the advantages of each radiolabel in clinical and investigative studies. Fourteen patients were included in this prospective study. Granulocytes were labelled with both 111In-tropolonate and 99mTc-HMPAO. In vitro shape change assay and in vivo distribution and recovery studies were performed to assess the activation of and damage to these cells due to the labelling procedure. The comparative kinetics of 111In and 99mTc in the blood, liver, spleen, and bone marrow were studied by blood sampling and dual radionuclide imaging early (1 h) and late (24 h) after injection. The functional integrity of the double-labelled granulocytes and the feasibility of the technique were investigated in 14 patients with a painful prosthetic hip due to causes other than infection. The efficiency of double labelling was 63% (SD 14%) for 111In and 39% (SD 12%) for 99mTc-HMPAO. In vitro granulocyte activation and ex vivo recovery values were comparable to those from single radionuclide labelling. No artefactual granulocyte sequestration was seen in the lungs or liver. The radioactivity was distributed between the liver, spleen and bone marrow and, to a lesser extent, the lung. Early 99mTc counts in the liver, spleen and bone marrow, in relation to background, were significantly higher than 111In counts while the reverse was seen in late images.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1991

Functional alterations of human platelets following indium-111 labelling using different incubation media and labelling agents.

Human platelets were labelled in the absence or presence of plasma using indium-111 labelled oxine sulphate, tropolone or 2-mercaptopyridine-N-oxide (MPO). Under in vitro and in vivo conditions, platelet functions were evaluated by measuring their aggregability, survival, recovery and early distribution. High labelling efficiency was achieved in saline labelling, whereas with plasma labelling, it was necessary to concentrate the platelet-rich plasma to 4.8 x 10(6) platelets/microliters. The aggregation of platelets labelled in plasma or saline was compared with that of controls; platelets labelled in saline showed lower aggregability in 2 microM ADP but not in 5 microM ADP nor with collagen. No significant differences in platelet survival and recovery were noted between platelets labelled in plasma and those labelled in saline. Our results indicate that partial loss of ADP aggregability in vitro does not influence the in vivo viability of platelets labelled in saline. Scintigraphic studies showed that platelets labelled in a saline medium were temporarily sequestrated in the liver but not in the spleen or heart. Thus, platelet labelling in saline does not affect platelet function adversely, but platelets labelled in plasma are more desirable for assessing the early distribution of platelets in the reticuloendothelial system.

Adult↗

PubMed2001

Improved beta-thujaplicin production in Cupressus lusitanica suspension cultures by fungal elicitor and methyl jasmonate.

Production of a novel antimicrobial tropolone, beta-thujaplicin, in Cupressus lusitanica suspension cultures was studied by using a variety of chemicals and fungal elicitors. Sodium alginate, chitin, and methyl jasmonate resulted in 2-, 2.5-, and 3-fold higher beta-thujaplicin production, respectively, than in the control. Significantly improved beta-thujaplicin production (187 mg l(-1)) was obtained using a high cell density (180-200 g l(-1)) and fungal elicitor treatment [10 mg (g fresh cells)(-1)] in a production medium with a high ferrous ion concentration (0.3 mM). This improved volumetric productivity was 3- to 4-fold higher than obtained under standard conditions. A synergistic effect of fungal elicitor and ferrous ion on beta-thujaplicin production was also suggested by our study. Plant cell culture technology is a promising alternative for producing a large variety of secondary metabolites that are widely used as food additives, pharmaceuticals, and dairy products (Verpoorte et al. 1999). Thus, beta-thujaplicin production by plant cell cultures was developed with the goal of commercial application (Berlin and Witte 1988; Itose and Sakai 1997; Ono et al. 1998). However, the production of beta-thujaplicin by plant cell cultures is still not competitive for use in industrial applications. In this study, we assessed the effects of methyl jasmonate, alginate, chitin, and fungal elicitor on beta-thujaplicin production; we obtained a significantly elevated beta-thujaplicin production by using an improved culture strategy.

Acetates↗

PubMed2006

Labelling of human mesenchymal stem cells with indium-111 for SPECT imaging: effect on cell proliferation and differentiation.

PURPOSE: Stem cell therapy seems to be a new treatment option within cardiac diseases to improve myocardial perfusion and function. However, the delivery and traceability of the cells represent a problem. Radioactive labelling with 111In could be a method for tracking mesenchymal stem cells (MSCs). However, 111In could influence the viability and differentiation capacity of MSCs, which would limit its use. Therefore, the aim of this study was to evaluate the influence of 111In labelling in doses relevant for SPECT imaging in humans on the viability and differentiation capacity of human MSCs. METHODS AND RESULTS: Human MSCs isolated from bone marrow were incubated with 111In-tropolone (15-800 Bq/cell). The labelling efficiency was approximately 25% with 30 Bq/cell 111In. The MSC doubling time was 1.04+/-0.1 days and was not influenced by 111In within the range 15-260 Bq/cell. Using 30 Bq 111In/cell it was possible to label MSCs to a level relevant for clinical scintigraphic use. With this dose, 111In had no effect on characteristic surface and intracellular markers of cultured MSCs analysed both by flow cytometry and by real-time polymerase chain reaction. Further, the labelled MSCs differentiated towards endothelial cells and formed vascular structures. CONCLUSION: It is possible to label human MSCs with 111In for scintigraphic tracking of stem cells delivered to the heart in clinical trials without affecting the viability and differentiation capacity of the MSCs. This creates an important tool for the control of stem cell delivery and dose response in clinical cardiovascular trials.

Cell Differentiation↗

PubMed2001

Abnormal platelet adhesion on abdominal vessels in asymptomatic patients with paroxysmal nocturnal hemoglobinuria.

Platelet kinetic studies were performed in eight patients with paroxysmal nocturnal hemoglobinuria (PNH) but without symptoms or signs related to thrombosis. Indium-111 tropolonate labeled autologous platelets were used to determine mean platelet life and platelet production rate; total body images were also acquired. Three of seven patients with a thrombocytopenia showed a shortened mean platelet life, and the other five demonstrated a (nearly) normal platelet survival. Four of the seven patients with thrombocytopenia showed a decreased platelet production rate, but bone marrow cellularity was normo- to hypercellular. These observations suggest ineffective thrombopoiesis in 57% of the patients with PNH and thrombocytopenia. Total body imaging was performed during the platelet kinetic study in order to study organ uptake. Imaging of the abdominal vessels was demonstrated in all patients, suggesting enhanced adherence of platelets to the endothelium in patients with PNH. Since thrombosis of especially the abdominal vessels is a major cause of morbidity and mortality, the use of antithrombotic drugs might be considered despite the absence of abdominal complaints.

Abdomen↗

PubMed1992

Aging of tubulin at neutral pH: stabilization by colchicine and its analogues.

The effect of colchicine and its analogues, allocolchicine, 2,3,4-trimethoxy-4'-carbomethoxy-1,1'biphenyl, 2,3,4,4'-tetramethoxy-1,1'-biphenyl, 2,3,4-trimethoxy-4'-acetyl-1,1'-biphenyl, and tropolone methyl ether, on the aging process of tubulin has been examined. In contrast to the vinca alkaloid drugs which accelerate the formation of the paucidisperse 9 S polymers by a factor of 3.5, the colchicine class of ligands stabilize alpha,beta-tubulin. Less than 10% of the protein is transformed into the aggregates after 50 h of incubation in the presence of 1 x 10(-3) M colchicine, as compared to nearly 70-75% transformation in its absence. These results are supported by fluorescence examination of the retention of colchicine binding ability, as well as circular dichroism spectroscopy. In the presence of colchicine, the rate determining step is a conformational change, just as in its absence. The colchicine analogues which bind to tubulin in a rapidly reversible equilibrium were almost as effective in tubulin stabilization. Addition of vincristine to the system reduced the stability of the tubulin-colchicine complex. Furthermore, vincristine was found to have the same effects on the fresh complex as it does on pure tubulin; i.e., it induced the isodesmic linear polymerization and inhibited assembly into the microtubule-mimicking large polymers. This inhibition, however, was stoichiometric, whereas it is substoichiometric in the case of microtubules.

Animals↗

PubMed1988

Preparation of liposomes entrapping a high specific activity of 111In3+-bound inulin.

Targeting liposomes to specific tissues or cells require the unequivocal determination of the uptake of liposomes at the cellular level. The present report describes the preparation of liposomes entrapping a high specific activity of 111In3+-bound inulin, and the potential applications of a multiple labeling technique for characterizing the extent of uptake of liposomes by tissues or different cells in a given tissue in vivo. The labeling method involves the application of the technique of acetylacetone-mediated, ionophoric loading of 111In3+ into liposomes entrapping an inulin derivative to which a strong chelating agent, diethylenetriamine-pentaacetic acid (DTPA), is bound. Subsequent ionophoric removal of the weakly bound 111In3+ by incubating the previously 111In3+-loaded liposomes with 10 mM nitrilotriacetic acid and 100 microM tropolone at room temperature for 20 min results in the preparation of liposomes entrapping 111In3+-DTPA-inulin. Our method of preparation yields net efficiencies of converting 63-78% of the externally added 111In3+ to liposome-entrapped 111In3+-DTPA-inulin.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1977

Electrically induced release of [3H]dopamine from slices obtained from different rat brain cortex regions. Evidence for a widespread dopaminergic innervation of the neocortex.

Slices obtained from the deeper layers of the rat dorsal frontal, parietal and occipital brain cortex were incubated in vitro with 6.25 X 10-7 M [3H]dopamine (DA), and subsequently superfused and electrically stimulated, while held on quick transfer electrodes, and changes in the efflux of 3H and of the individual amines measured. The separation of the amines, with quantitative recoveries, was performed by chromatography on cation-exchange resins eluted sequentially with water, 1 N HCL AND 6 M urea i 1 N HCl. When no drugs were used, the prestimulation efflux was entirely formed by deaminated metabolites, while following stimulation there was an increase in the efflux of deaminated metabolites, and considerable amounts of [3H]-noradrenaline (NA) now appeared. No DA was present in the pre- or poststimulation medium. Similar results were obtained in all the regions studied. When the slices were incubated with 10-5 M desmethylimipramine (DMI), 10-4 Mnialamide and 10-4 M tropolone, before and during incubation with [3H]DA, it was observed that, prior to stimulation, the efflux was composed of deaminated metabolites, DA and 3-methoxytyramine (MTA), and following the electrical stimulus there was an increased release of DA, NA and deaminated compounds (in order of decreasing release), while no change in that of MTA was evident. The stimulus-induced release of DA was greatest from frontal slices, intermediate from parietal, and lowest from occipital ones. DMI-resistant uptake of [3H]DA also diminished when passing from frontal to occipital. These findings are interpreted as due to the presence of dopaminergic axon terminals in all the regions studied, but with a density that diminishes in a rostrocaudal direction.

Adrenergic Fibers↗

PubMed1976

Turnover of acid dopamine metabolites in striatal and mesolimbic tissue of the rat brain.

The subject of this study was the disappearance of 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) from the brain of rats treated with pargyline and pargyline in combination with tropolone. The DOPAC decline was exponential after pargyline treatment, while the HVA decline became a first order elimination when in addition to monoamine oxidase, catechol-O-methyl transferase was also inhibited. The turnover of the metabolites was calculated by multiplying the steady state level by the fractional rate constant (k). The DOPAC turnover was found to be 23.3 nmol/g/h in the corpus striatum and 22.6 nmol/g/h in the mesolimbic structures. The HVA turnover was 11.2 nmol/g/h in the corpus striatum and 6.7 nmol/g/h in the mesolimbic structures. The data showed that under control conditions DOPAC is only partially O-methylated to HVA, while the formation of HVA via the methoxytyramine pathway is unlikely. DOPAC turnover therefore probably approximates dopamine turnover.

3,4-Dihydroxyphenylacetic Acid↗