SEARCH · Search PubMed
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Synthesis of 4-aminotropones through a cyclization-ring expansion process occurs in a single step and with excellent yields from 4-amino-2,5-cyclohexadienones (p-quinamines) bearing a 4-sulfinyl or sulfonyl methyl group.
The 20 cysteine residues of tubulin are heterogeneously distributed throughout its three-dimensional structure. In the present work, we have used the reactivity of these cysteine residues with 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a probe to detect the global conformational changes of tubulin under different experimental conditions. The 20 sulfhydryl groups can be classified into two categories: fast and slow reacting. Colchicine binding causes a dramatic decrease in the reactivity of the cysteine residues and causes complete protection of 1.4 cysteine residues. Similarly, other colchicine analogs that bind reversibly initially decrease the rate of reaction; but unlike colchicine they do not cause complete protection of any sulfhydryl groups. Interestingly, in all cases we find that all the slow reacting sulfhydryl groups are affected to the same extent, that is, have a single rate constant. Glycerol has a major inhibitory effect on all these slow reacting sulfhydryls, suggesting that the reaction of slow reacting cysteines takes place from an open state at equilibrium with the native. Ageing of tubulin at 37 degrees C leads to loss of self-assembly and colchicine binding activity. Using DTNB kinetics, we have shown that ageing leads to complete protection of some of the sulfhydryl groups and increased reaction rate for other slow reacting sulfhydryl groups. Ageing at 37 degrees C also causes aggregation of tubulin as indicated by HPLC analysis. The protection of some sulfhydryl groups may be a consequence of aggregation, whereas the increased rate of reaction of other slow reacting sulfhydryls may be a result of changes in global dynamics. CD spectra and acrylamide quenching support such a notion. Binding of 8-anilino-1-naphthalenesulfonate (ANS) and bis-ANS by tubulin cause complete protection of some cysteine residues as indicated by the DTNB reaction, but has little effect on the other slow reacting cysteines, suggesting local effects.
Sunburn cells are thought to represent ultraviolet B-induced apoptotic keratinocytes. It has been demonstrated that enzymatic and nonenzymatic antioxidants effectively suppress sunburn cell formation, indicating that reactive oxygen species may play a role in the progression of ultraviolet B-induced apoptosis. Metallothionein, a cytosol protein, has antioxidant activity, and overexpression of metallothionein has been reported to reduce the number of sunburn cells in mouse skin. We have also demonstrated that overexpression of metallothionein inhibits ultraviolet B-induced DNA ladder formation in mouse keratinocytes. These findings support the hypothesis that cellular metallothionein may play an important role in the inhibition of ultraviolet B-induced apoptosis in keratinocytes through its antioxidant activity. In the present study, we investigated the effects of beta-thujaplicin, an extract from the woods of Thuja plicata D. Don. and Chamaecyparis obtuse, Sieb. et Zucc., on ultraviolet B-induced apoptosis in keratinocytes and on metallothionein induction. Topical application of beta-thujaplicin decreased the number of ultraviolet B-mediated sunburn cells and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling-positive cells in mouse ear skin. Incubation with beta-thujaplicin suppressed ultraviolet B-induced DNA ladder formation in cultured mouse keratinocytes. Histochemical analysis showed that topical application of beta-thujaplicin induced metallothionein protein in mouse skin. Northern analysis and western blotting revealed significant induction of metallothionein mRNA and metallothionein protein, respectively, in beta-thujaplicin-treated cultured mouse keratinocytes. These findings indicate that beta-thujaplicin inhibits ultraviolet B-induced apoptosis in keratinocytes and strongly suggest that the inhibitory mechanism is due to the antioxidant activity of metallothionein induced by the agent.
The explosive expansion of knowledge in immunology in recent decades has already affected the research and practice of nuclear medicine in several ways. New hematopoietic cells have been isolated and their functions discovered, including hematopoietic stem cells and dendritic cells (DCs). Many new humeral factors have been found that have potent effects on cells, including cytokines, growth factors, and specialized proteins. Radiolabeled compounds are needed to follow the pharmacodynamics of the humeral factors and to follow the migration of mobile cells in animals and humans. In this article, only DCs, cytokines, and growth factors used clinically are discussed. DCs are essential for defense against infectious diseases. Autologous DCs cultured for a week and pulsed with tumor antigens have already proved highly immunogenic compared with other methods for activating cytotoxic T cells, and preliminary studies suggest that DCs are more potent for tumor cell killing than monoclonal antibodies. DCs, unfortunately, also play an important role in causing certain human diseases. In allograft transplants, residual donor DCs are responsible for the cellular rejection; if they could be eliminated, rejection could be prevented. These cells are also detrimental in rheumatoid arthritis, other autoimmune diseases, asthma, and chronic obstructive pulmonary disease. Cytokines such as interleukin-2 and such growth factors as granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor, administered to patients with malignancies to alleviate the leukopenia of chemotherapy agents, frequently alter the tissue distribution of radiopharmaceuticals; these alterations may be confused with disease.
Antiplasmodial and cytotoxicity testing of five highly oxygenated natural products (6R,12R,14R-colletoketol, 6R,11R,12R,14R-colletoketodiol, dihydrobotrydial, pycnidione, and 3R,4S-hydroxymellein), all derived from fungi of marine origin, showed one of them, pycnidione, to have activities against three different strains of Plasmodium falciparum in the sub-micromolar (microM) range. Although the mean selectivity index of 1 for the observed antiplasmodial activity of 4 is low, pycnidione's usefulness as a potential lead structure should not be ignored.
The role of microtubules in 3H-labeled protein secretion in rat lacrimal glands was probed by the use of colchicine and two of its analogues that reversibly bind to tubulin. These analogues were 2-methoxy-5-(2,3,4,4'-trimethoxyphenyl)-2,4,6-cycloheptatriene-1-o ne and 2,3,4,4'-tetramethoxy-1,1'-biphenyl, the latter having been synthesized for these studies. Immunofluorescence revealed that untreated exocrine acinar cells contained an intact microtubule network, which was totally abolished by drug addition. Subsequent drug removal restored the network for the two reversibly binding drugs--more rapidly so for the biphenyl, but this was not the case with colchicine. The protein-secretory process was examined by adding the three drugs at various stages--prepulse incubation, pulse, maturation, apical storage of granules, and discharge under cholinergic stimulation. Comparison with the kinetics of microtubular network restoration, which differed for the two reversibly binding drugs, led to the conclusion that the microtubular system is critical to the maturation phase of secretion.
The biosynthesis of a phytoalexin, beta-thujaplicin, in Cupressus lusitanica cell cultures can be stimulated by a yeast elicitor, H(2)O(2), or methyl jasmonate. Lipoxygenase activity was also stimulated by these treatments, suggesting that the oxidative burst and jasmonate pathway may mediate the elicitor-induced accumulation of beta-thujaplicin. The elicitor signalling pathway involved in beta-thujaplicin induction was further investigated using pharmacological and biochemical approaches. Treatment of the cells with calcium ionophore A23187 alone stimulated the production of beta-thujaplicin. A23187 also enhanced the elicitor-induced production of beta-thujaplicin. EGTA, LaCl(3), and verapamil pretreatments partially blocked A23187- or yeast elicitor-induced accumulation of beta-thujaplicin. These results suggest that Ca(2+) influx is required for elicitor-induced production of beta-thujaplicin. Treatment of cell cultures with mastoparan, melittin or cholera toxin alone or in combination with the elicitor stimulated the production of beta-thujaplicin or enhanced the elicitor-induced production of beta-thujaplicin. The G-protein inhibitor suramin inhibited the elicitor-induced production of beta-thujaplicin, suggesting that receptor-coupled G-proteins are likely to be involved in the elicitor-induced biosynthesis of beta-thujaplicin. Indeed, both GTP-binding activity and GTPase activity of the plasma membrane were stimulated by elicitor, and suramin and cholera toxin affected G-protein activities. In addition, all inhibitors of G-proteins and Ca(2+) flux suppressed elicitor-induced increases in lipoxygenase activity whereas activators of G-proteins and the Ca(2+) signalling pathway increased lipoxygenase activity. These observations suggest that Ca(2+) and G-proteins may mediate elicitor signals to the jasmonate pathway, and the jasmonate signalling pathway may then lead to the production of beta-thujaplicin.
Roles of jasmonate and ethylene signalling and their interaction in yeast elicitor-induced biosynthesis of a phytoalexin, beta-thujaplicin, were investigated in Cupressus lusitanica cell cultures. Yeast elicitor, methyl jasmonate, and ethylene all induce the production of beta-thujaplicin. Elicitor also stimulates the biosynthesis of jasmonate and ethylene before the induction of beta-thujaplicin accumulation. The elicitor-induced beta-thujaplicin accumulation can be partly blocked by inhibitors of jasmonate and ethylene biosynthesis or signal transduction. These results indicate that the jasmonate and ethylene signalling pathways are integral parts of the elicitor signal transduction leading to beta-thujaplicin accumulation. Methyl jasmonate treatment can induce ethylene production, whereas ethylene does not induce jasmonate biosynthesis; methyl jasmonate-induced beta-thujaplicin accumulation can be partly blocked by inhibitors of ethylene biosynthesis and signalling, while blocking jasmonate biosynthesis inhibits almost all ethylene-induced beta-thujaplicin accumulation. These results indicate that the ethylene and jasmonate pathways interact in mediating beta-thujaplicin production, with the jasmonate pathway working as a main control and the ethylene pathway as a fine modulator for beta-thujaplicin accumulation. Both the ethylene and jasmonate signalling pathways can be regulated upstream by Ca(2+). Ca(2+) influx negatively regulates ethylene production, and differentially regulates elicitor- or methyl jasmonate-stimulated ethylene production.
With 111In-labeled platelets and 125I-labeled bovine fibrinogen, regional mapping of platelet and fibrinogen deposition on leaflets and sewing rings was obtained. Ten Holstein calves received 25-mm mitral valves (ISLM) and were killed 1, 14, and 30 days after implantation. Twenty-four hours before the calves were killed, 350 to 450 microCi of 111In-labeled platelets and 200 to 250 microCi of 125I-labeled bovine fibrinogen were administered intravenously. The components of the tissue valves, i.e., three leaflets and sewing rings, were separated. Each leaflet was cut into four sections: free edge, central zone, flexion zone, and attachment zone. From the radioactivity in blood, leaflet zones, sewing rings, area of leaflet zones, platelet count, and fibrinogen level in blood, the mean regional density of adherent platelets, fibrinogen-fibrin, and fibrinogen/platelet were calculated. The density of platelets and fibrinogen deposited on the components of the valves decreases with time postimplantation. The number of fibrinogen molecules per platelet is fivefold to 20-fold higher than that of the receptor concentration on platelets on leaflet zones, suggesting the heterogeneity of fibrinogen-fibrin in thrombus and components of the valve.
Platelet consumption in a hollow-fiber membrane oxygenator (HFMO) and arterial filter (AF) during cardiopulmonary bypass (CPB) was quantified in five pigs using Indium-111 labeled autologous platelets. Platelet labeling was performed 20-24 hours before CPB. After general endotracheal anesthesia, the pigs were systemically heparinized and were placed on CPB via a median sternotomy. After 3 hours of CPB, radioactivity was quantified with a gamma camera and an ionization chamber. The percent of injected dose (mean +/- SD) was 0.79 +/- 0.45 in the HFMO, 2.52 +/- 0.93 in AF, 4.3 +/- 1.2 in blood loss during CPB. Platelet consumption in HFMO during CPB was lower than in bubble oxygenators (19%) or silicone membrane oxygenators (12%) as observed in previous studies.
A patient with active thrombotic thrombocytopenic purpura underwent examination with In-111 troponolate labeled autologous platelets. A mean platelet survival of 1.2 days was found, comparable to formerly described studies using other methods. Furthermore, remarkable renal uptake of radioactivity consistent with abnormal platelet trapping was demonstrated. In the absence of any other signs of renal involvement, the conclusion was reached that the platelet scan is a sensitive and direct method for showing renal involvement in thrombotic thrombocytopenic purpura.
Radiolabeled blood cells are widely used for both clinical and research studies. In vitro and in vivo studies have shown the tagging process does not significantly affect function. The labeling techniques and clinical uses of labeled leukocytes and platelets are reviewed.
The kinetics of human autologous granulocytes, separated and labelled with 111In without isolation from plasma, have been studied in subjects with and without sepsis with the aim of identifying the fate and sites of destruction of granulocytes in man. In subjects without inflammatory disease, 111In granulocyte recovery in faeces, urine and saliva over 4 d was less than 1% of the dose, so that the activity visualized by the gamma camera represented almost 100% of the dose. On images taken at 24 and 48 h, this activity was distributed between spleen, bone marrow and liver, with foci of additional abnormal activity in subjects with inflammatory disease. Splenic activity fell between 40 min and 24 h, consistent with the presence of a splenic granulocyte pool, but remained constant after 24 h. Since granulocyte clearance from the blood was predominantly completed by 24 h, the residual splenic activity at that time reflected splenic granulocyte destruction. In patients with sepsis, the fall in splenic activity was greater than in those without, implying diversion of granulocytes from splenic destruction to tissue utilization when inflammation is present. Bone marrow activity increased between 40 min and 24 h and then remained stable. Granulocytes that were extensively manipulated in saline prior to labelling failed to localize in marrow, suggesting that visualization of the latter reflected destruction of intact, normal granulocytes. Although the changes in splenic and marrow activities terminated at 24 h, at which time granulocyte clearance from blood was at least 80% completed, plasma 111In remained essentially unchanged between 40 min and 48 h at less than 5% of the dose, discounting it as the source of splenic and marrow activities.
Major surgery evokes an endocrine stress response, characterized by increased serum cortisol, plasma adrenaline and noradrenaline. Furthermore, surgical stress is accompanied by lymphopenia and granulocytosis in peripheral blood. The changes in peripheral white blood cells have been demonstrated after surgery as well as after cortisol infusion. The aim of the present study was to investigate to which tissues/organs peripheral blood lymphocytes are redistributed after major surgery. From 20 rabbits lymphocytes were isolated from peripheral blood, labelled with indium-111-tropolene and reinjected intravenously into the rabbits. Ten of the rabbits underwent major surgery (upper laparatomy) during general anaesthesia, while the control group (n = 10) was anaesthetized without surgery. The endocrine stress response to surgery was measured as serum cortisol, plasma adrenaline and noradrenaline. The redistribution of lymphocytes was imaged with a gamma camera and calculated with a connected computer before, 2, 4, and 7 h after the skin incision. Compared to preoperative values, laparotomy resulted in an increase in serum cortisol from 116.6 to 461.9 nmol/l (mean) and a decrease in the fraction/percentage of lymphocytes in peripheral blood from 43.8% to 14.7% 7 h after surgery. Simultaneously, the activity of the heart and lungs together decreased to 76.1% of initial values, while the spleen activity was unaffected. The radioactivity of the lymphatic tissue increased to 137.8% and 134.7%, respectively, 4 and 7 h after the start of surgery. The results indicate that major surgery induces a redistribution of lymphocytes from peripheral blood to lymphatic tissue. It is suggested that the endocrine stress response may be of major importance.
Tubulin exists as various isoforms, which differ in their assembly, drug-binding properties, and the dynamic properties of the microtubules they compose. One of the most striking differences in drug binding among the isoforms is observed with colchicine, which binds much better to the alphabeta(II) and alphabeta(IV) isoforms than to the alphabeta(III) isoform. Here we have studied the interaction of these isoforms with 2-methoxy-5-(2',3',4'-trimethoxyphenyl) tropone (MTPT), an analog of colchicine that lacks the B-ring. The kinetics of association and dissociation were studied fluorometrically, and the kinetic parameters for the two-step binding were determined for different beta-tubulin isoforms. The apparent on-rate constants for alphabeta(II), alphabeta(III) and alphabeta(IV) were 13358, 4558 and 10828 M(-1) s(-1), the off-rate constants (k(-2)) were 0.04, 0.03 and 0.02 s(-1), and the affinity constants are 3.33 x 10(5), 1.56 x 10(5) and 5.44 x 10(5) M(-1), respectively. The differences in kinetic parameters among different beta-tubulin isoforms are greatly reduced when the B-ring is removed. Our results indicate that the B-ring plays a major role in determining the isoform differences, and the results might be of importance for designing tissue-specific analogs of colchicine for cancer chemotherapy.
The relationship between phenolsulfotransferase (PST) and catechol-O-methyltransferase (COMT) in the metabolism of free 3,4-dihydroxyphenylethylamine (DA, dopamine) in the rat brain was studied. In rats not pretreated with a monoamine oxidase (MAO) inhibitor a huge increase of free DA in the brain, following an intraperitoneal injection of L-3,4-dihydroxyphenylalanine (L-DOPA) or an intraventricular injection of free DA, did not lead to any noticeable change in DA sulfate or 3-methoxytyramine (3-MT), which remained undetectable by the present HPLC method. However, in rats previously treated with the MAO inhibitors pargyline or tranylcypromine, the same L-DOPA or free DA treatment resulted in significant increases in both 3-MT and DA sulfate in the hypothalamus, brainstem, and striatum. This response of COMT and PST was not affected by prior treatment of the rats with 6-hydroxydopamine, which suggests that O-methylation and sulfoconjugation occur outside adrenergic neurons not destroyed by the neurotoxin. Inhibition of COMT activity did not lead to any increase in DA sulfate, which showed that despite their common mode of action (both enzymes react preferentially at the same hydroxyl group in the DA molecule), the two enzymes are not competitive. After MAO inhibition there were strong correlations between an increase in DA sulfate and 3-MT on the one hand, and between free DA and 3-MT on the other. Because 3-MT is a marker of central DA release, these data suggest that inhibition of MAO activity not only affects DA metabolism by this enzyme but also influences DA release in the rat brain.
1 The Falck-Hillarp histochemical technique was used to locate extraneuronal sites of accumulation of isoprenaline in trachea and atria from guinea-pig and cat. With a tissue exposure time to formaldehyde gas of 3 h, isoprenaline was located as green fluorescence.2 Quantitative microphotometry was used to measure fluorescence intensity within cells in the trachealis smooth muscle and the atrial myocardium of both species.3 After incubation of tissues in 50 muM isoprenaline, specific fluorescence was seen in trachealis smooth muscle of both species and in the atrial myocardium of cat but not guinea-pig. In both species, fluorescence was also seen in the chondroblasts of the tracheal cartilage and in blood vessels in all tissues.4 In trachealis smooth muscle of both species and in cat atrial myocardium, fluorescence brightness, resulting from incubation of tissues in 50 muM isoprenaline was significantly increased by 200 muM beta-thujaplicin, an inhibitor of catechol-O-methyl transferase (COMT). In the presence of beta-thujaplicin, fluorescence was not visible in guinea-pig atrial myocardium with 50 muM isoprenaline, although fluorescence brightness measured in myocardial cells was now greater than that in corresponding controls.5 The fluorescence intensity seen in cat and guinea-pig trachealis smooth muscle cells and in cat atrial myocardial cells after incubation in 50 muM isoprenaline was decreased significantly in the presence of phenoxybenzamine (100 muM). In guinea-pig atria, phenoxybenzamine had no effect on myocardial fluorescence. Fluorescence intensity was also decreased if the incubation with isoprenaline was carried out at 0 degrees C or if the post-incubation washing temperature was 37 degrees C instead of 0 to 2 degrees C.6 The results demonstrate that the fluorescence histochemical technique can be used to locate isoprenaline in tissues. They also indicate that guinea-pig and cat trachealis smooth muscle cells and cat atrial myocardial cells can accumulate isoprenaline (a) by a mechanism sensitive to phenoxybenzamine and (b) into sites in which COMT plays a functional role in inactivating isoprenaline at the concentration used in these histochemical experiments (50 muM). In contrast, the guinea-pig atrial myocardial cells may have a minimal capacity to accumulate isoprenaline by a phenoxybenzamine-sensitive uptake mechanism.
To evaluate the thrombogenicity of transvenous silicone and polyurethane pacemaker leads, 9 of 12 anesthetized Yorkshire pigs (27-32 kg) were implanted with silicone (n = 5) or polyurethane (n = 4) pacemaker leads via a femoral vein. The remaining three pigs served as controls. All 12 pigs were injected with autologous indium-111 labeled platelets (300-420 muCi) 24 hours before anesthesia induction. The pigs were monitored for 3 hours under a gamma camera. Radioactivity in blood and lead segments was measured with a gamma counter. Platelet deposits were denser on silicone leads (441.58 +/- 915.0 to 2.19 +/- 2.07) than on polyurethane leads (1.21 +/- 1.33 to 0.27 +/- 0.14) (P > 0.05). Denser platelet deposits were detected at the tip of all leads. Density of platelet deposits declined from tip to distal segments in silicone leads. The percentage of injected platelet radioactivity in the lungs of pigs with either silastic leads (12.9 +/- 2.3%) of polyurethane leads (10.1 +/- 2.2%) was higher than in the controls (4.6 +/- 0.5%) (P < 0.05). This difference indicates thrombus formation and embolization in the lungs early after lead implantation. Thrombogenicity of polyurethane leads may be lower than that of silicone leads.