Binding of 67 Ga and 59 Fe to ferritin or transferrin

The bindings of 67Ga and 59Fe to ferritin or transferrin in vitro has been investigated. Affinity constants have been measured using the equilibrium dialysis, and the results have been obtained as follows: 1 Apo-ferritin could not bind to 67Ga until it was transformed into ferritin in presence of Fe-citrate. On the contrary, the affinity of 67Ga to ferritin was reduced when Fe was released from ferritin; thus indicating that Fe-core has been required for the binding of 67Ga to ferritin. 2 Binding of 67Ga to ferritin was inhibited with apo-transferrin, and this was also shown in the case of 59Fe. In the presence of NaHCO3 or citrate, more remarkable inhibitions were observed. NaHCO3 or citrate was found to give a synergistic effect on the binding of 67Ga to transferrin, as well as Fe-transferrin. Therefore, both 67Ga and 59Fe could not bind to ferritin in the state of 67Ga- or 59Fe-transferrin. 3 The release of 59Fe from 59Fe-transferrin was enhanced with adenosine triphosphate (ATP), citrate, or ascorbic acid, while any of these reagents did not affect the release of 67Ga from 67Ga-transferrin. The comparison of 59Fe and 67Ga through their bindings to ferritin or transferrin has suggested one of points to distinguish 67Ga from 59Fe in the cell.     

Relation between 67Ga uptake and the stage of inflammation induced by turpentine oil in rats

For the establishment of the experimental system to judge easily the effect of anti-inflammatory drug, 67Ga-citrate was used. The weight of granuloma tissues induced by inflammable agent, turpentine oil, gradually increased and reached a maximum at 6 days after the administration of turpentine oil. Gallium-67 accumulation in the inflammatory lesions showed also a maximum at 6 days after that. Both patterns were closely similar each other. These results showed that the processes and/or stages of inflammation could be indicated by the pattern of 67Ga uptake.     

Interactions between59Fe(III)-polyphosphates and clayey meadow soil

Interaction between iron(III)-diphosphate and iron(III)-triphosphate and Ca-form of a clayey meadow soil was followed over a period of three days using radiotracer technique and kinetic evaluation of the results performed.⁵⁹Fe served to determine the quantity of iron,⁴⁵Ca to measure the calcium, and phosphorus was measured spectrophotometrically. Approximately 80% of both iron chelates disappeared from the solution during the time of the experiment as a result of two well distinguishable reactions. One of them is a rapid interfacial process of about 10 minutes and the other is a slow reaction leading to the decomposition of iron(III)-polyphosphate chelates. The two processes could be separated using the Christiansen equation.     

The influences of dexamethasone and indomethacin on 67Ga uptake by normal tissues

Dexamethasone (DEX) increased 67Ga uptake by the liver and spleen at 4, 8, and even 24 h after the injection of 67Ga. These results showed that DEX influenced 67Ga accumulation as well as the initial entry of 67Ga in the liver and spleen. On the other hand, indomethacin (IM) decreased 67Ga uptake by the liver and spleen at 4 h after the injection of 67Ga but did not influence the uptake at 8 or 24 h after the injection of 67Ga. Moreover, DEX or IM little influenced 67Ga uptake by the kidney and muscle. These results suggest that the influence of DEX or IM on 67Ga uptake or accumulation is specific for the liver and spleen.     

Subcellular detection and localization of the drug transporter P-glycoprotein in cultured tumor cells

In vitro studies on the cellular location of P-glycoprotein (Pgp) are reported with the aim to clarify the relationship between its intracellular expression and the multidrug resistance (MDR) level of tumor cells. Pgp was found abnormally expressed on the plasma membrane of tumor cells with "classical" MDR phenotype. However, Pgp was also often detected on the nuclear envelope and on the membrane of cytoplasmic organelles. The hypothesis that this drug pump maintains a transport function when located in these compartments, is still under debating. Our results, together with those obtained by other researchers, demonstrate that cytoplasmic Pgp regulates the intracellular traffic of drugs so that they are no more able to reach their cellular targets. In particular, we revealed that in MDR breast cancer cells (MCF-7) a significant level of Pgp was expressed in the Golgi apparatus. A similar result was found in human melanoma cell lines, which never undergone cytotoxic drug treatment and did not express the transporter molecule on the plasma membrane. A strict relationship between intracellular Pgp and intrinsic resistance was demonstrated in a human colon carcinoma (LoVo) clone, which did not express the drug transporter on the plasma membrane. Finally, a structural and functional association between Pgp and ERM proteins has been discovered in drug-resistant human T- lymphobastoid cells (CEM-VBL 100). Our findings strongly suggest a pivotal role of the intracytoplasmic Pgp in the transport of drugs into cytoplasmic vesicles, thus actively contributing to their sequestration and transport outwards the cells. Thus, intracellular Pgp seems to represent a complementary protective mechanism of tumor cells against cytotoxic agents.     

Study on suppressed uptake by the liver in 67Ga scintigraphy

During the past 3 years, we have performed 1525 studies of 67Ga scintigraphy and 38 cases of these showed suppressed liver uptake; 19 cases after chemotherapy, 13 cases with liver dysfunction and 6 cases with competitive blockade. Many cases in chemotherapy used cyclophosphamide and vincristine, and were performed 67Ga scan within 1 week after chemotherapy. In cases showed suppressed uptake by liver after chemotherapy, serum Fe was markedly increased and UIBC was markedly decreased (p less than 0.01). This suggested that serum Fe influenced suppressed uptake by liver more than carrier-protein, disturbed hepatic cells, and receptors.     

New method for simultaneous determination of55Fe and59Fe in blood samples

A new simple method for the simultaneous determination of⁵⁹Fe and⁵⁵Fe concentration in 5 ml samples of blood is described and carefully evaluated. Before the measurement of the activity of the radionuclides, organic matter was eliminated by HNO₃?HClO₄ wet ashing. Iron was electroplated onto a copper plate. The accuracy of results was studied by assessing samples, which contained known amounts of radioactivity and determining the counts per nanocurie in each case. The accuracy of the results of⁵⁹Fe and⁵⁵Fe determinations was found to be about 5%.     

Interactions between59Fe (14C)EDTA and soils containing calcium carbonate

Interaction between FeEDTA and calcareous soils was followed over a period of four weeks using a radiotracer technique, and a kinetic evaluation of the results was performed.⁵⁹Fe served to determine the quantity of iron,¹⁴C to assay for EDTA and⁴⁵Ca to measure calcium. During the experiment, i. e. within four weeks in case of the chernozem soil 61% and in case of the clayey meadow soil 51% of the iron chelate dissapeared from the solution. The loss in soluble iron was partly due to a rapid sorption process of about an hour and partly due to the slow decomposition of FeEDTA to Fe(OH)₃. The two processes could be separated using the Christiansen equation.     

Comparative pharmacokinetics of 67Ga among the different routes of injection

The pharmacokinetic study of 67Ga-citrate (67Ga) following intravenous (i.v.), subcutaneous (s.c.) and intraperitoneal (i.p.) injection was performed in anesthetized rats using the repeated blood sampling method by cannulation technique into the external jugular vein. The disappearance of 67Ga from the blood following i.v. and s.c. injection was best fit a three-exponential equation. There was no significant difference between the areas under the curves following i.v. and s.c. injection of 67Ga. In the case of i.p. injection, the disappearance of 67Ga from the blood was described by a two-exponential equation. However, the maximum blood radioactivity was very low, and the disappearance rate of 67Ga from the blood was extremely slow compared to the other routes of injection. The conclusion from these results was that s.c. injection was as suitable as i.v. injection, but i.p. injection was not appropriate for the distribution study of 67Ga such as scintigraphy or autoradiography. However, i.p. route may be available for a special experiment which needs the long-time retention of 67Ga in the blood.     

Effect of sodium citrate on the blood disappearance of 67Ga

The effect of sodium citrate on the blood disappearance of 67Ga was examined in rats. The half life value of the alpha phase and the initial AUC value (0-60 min) were dose-dependently decreased by sodium citrate. The binding of 67Ga to serum proteins was also dose-dependently inhibited by sodium citrate.     

Preparation and biodistribution of [67Ga]-DTPA-rituximab in normal rats

Rituximab was successively labeled with [⁶⁷Ga]-gallium chloride after residulation with freshly prepared cyclic DTPA-dianhydride. The best results of the conjugation were obtained by the addition of 1 ml of a rituximab pharmaceutical solution (5 mg/ml, in phosphate buffer, pH 8) to a glass tube pre-coated with DTPA-dianhydride (0.01 mg) at 25 °C with continuous mild stirring for 30 minutes. Radio thin-layer chromatography showed an overall radiochemical purity of 96–99% at optimized conditions (specific activity = 300–500 MBq/mg, labeling efficiency 77%). The final isotonic ⁶⁷Ga-DTPA-rituximab complex was checked by gel electrophoresis for radiolysis control. Radio-TLC was performed to ensure the formation of only one species, followed by filtration through a 0.22 μm filter. Preliminary in vivo studies in normal rat model was performed to determine the biodistribution of the radioimmunoconjugate up to 6 hours.     

Preparation and biodistribution of [67Ga]-DTPA-gonadorelin in normal rats

Gonadorelin was successively labeled with [⁶⁷Ga]-gallium chloride after residulation with freshly prepared cyclic DTPA-dianhydride. The best results of the conjugation were obtained by the addition of 1 mg of a gonadorelin to a glass tube pre-coated with DTPA-dianhydride (0.33 mg) at 25 °C with continuous mild stirring for 1 hour. Radio thin layer chromatography showed an overall radiochemical purity of >90% at optimized conditions after labeling. HPLC showed a radiochemical purity more than 95% (specific activity = 400–450 GBq/M). The stability of the radioconjugate was tested in presence of human serum at 37 °C. Preliminary in vivo studies in normal rats were performed to determine the biodistribution of the conjugate up to 48 hours. The breast and ovaries uptakes were significantly high in first 15-minute post injection which is in agreement with the other reports regarding the presence of specific GnRH receptors. This tracer can be used in detection of GnRH receptor biodistribution in various diseases and malignancies.     

Gold-catalyzed intermolecular hydroalkoxylation of allenes; difference in mechanism between hydroalkoxylation and hydroamination

A wide range of alcohols 2 react with various allenes 1 in the presence of ClAuPPh₃/AgOTf catalyst at ambient temperature without solvent to produce allylic ethers 3. Contrary to the hydroamination, which proceeds through high chiral-face selectivity for chiral allenes to give the corresponding chiral allylic amines, transfer of chirality is not observed in the hydroalkoxylation, suggesting that the mechanism of the gold-catalyzed hydroalkoxylation is different from that of hydroamination.     

Uptake of 67Ga into the rat liver after treatment with thioacetamide

67Ga uptake of the liver began to elevate from the 1st day and reached a maximum at the 2nd day of treatment with thioacetamide (TIAA). Incorporation of 3H-thymidine into the liver DNA fraction was reached a maximum at the 1.5th day, and the value was 5.7 times of the control. The uronic acid content and 35S incorporation in the 1.2 M NaCl-soluble fraction which contained predominantly heparan sulfate (HS), were both peaked at the 2nd day. These patterns were in good agreement with that of 67Ga uptakes in the liver treated with TIAA. Pretreatment of aminoacetonitrile, an inhibitor of fibrosis, was effective in lowering the elevated uptake of 67Ga in TIAA-treated rat liver. Uptake of the 67Ga in the TIAA-treated liver was also inhibited when they were treated with cycloheximide, an inhibitor of protein synthesis. On the other hand no significant inhibition was observed in the cytosine arabinoside-treated-TIAA rats. These results suggest that HS may be involved in the 67Ga uptake in damaged liver, and that relation between 67Ga uptake and cell proliferation is secondary.     

On the67Ga-binding acid mucopolysaccharide in malignant tumor

The present study was undertaken to determine the structure of⁶⁷Ga-binding acid mucopolysaccharide in tumor tissues. It was determined from measuring neutral saccharide in the structure that the principal⁶⁷Ga-binding acid mucopolysaccharide in the tumor was keratan sulfate and/or keratan polysulfate. On the other hand, it was clarified from the results of mucopolysaccharase treatment that the main⁶⁷Ga-binding acid mucopolysaccharide in tumor was not keratan sulfate, heparan sulfate, heparin, nor chondroitin sulfate A, B, or C. Based on the present results, it was deduced that the main⁶⁷Ga-binding acid mucopolysaccharide in tumor is keratan polysulfate and that this acid mucopolysaccharide plays the most important role in tumor accumuation of⁶⁷Ga.     

The expression and localization of surface neoantigens in transformed and untransformed cultured cells infected with avian tumor viruses.

The presence and localization of neoantigens induced in cultured cells, infected or transformed with avian tumor viruses (ATV), were studied ultrastructurally on carbon platinum replicas of cell surfaces. The use of antibody, labeled with hemocyanin molecules, provided sensitive detection and analysis of cell surface antigen distribution. The subgroup-specific antigens of the viral envelope were found in considerable amount in the plasma membranes of ATV-infected chick embryo fibroblasts. The distribution of these antigens over the cell surface, evaluated on cells which were prefixed with glutaraldehyde, was found to be diffuse with a greater density on the cell processes in some cells. Reaction of antibody to viral envelope antigens with living ATV-infected cells resulted in a number of patterns of redistribution of membrane antigen-antibody complexes (AAC). Redistribution occurred in symmetrical or asymmetrical modes. The former consisted of randomly oriented aggregates (patches) of AAC over the cell surface. The latter included: (a) linear accumulation of AAC at cell margins; and (b) condensation of compexes into one or more centers of coalescence. These observations could be made on chick embryo cells infected (but not transformed) by avian leukosis virus, or on cells oncogenically transformed by avian sarcoma virus. The regions of coalescence were suggestive of the "capping" phenomenon seen in other systems, and their formation was temporally correlated with endocytosis of labeled AAC and the gradual loss of AAC from the surface. The effects of several biologically perturbing substances on the processes of redistribution were investigated in ALV-infected fibroblasts. Sodium azide, puromycin, actinomycin D, and colchicine had no effect on either form of asymmetrical redistribution. Cytochalasin B (CB) and iodoacetic acid (IAA) appeared to have some effect on the marginal redistribution, and to completely prevent the condensation into foci of coalescence (FC). When treated with these compounds, reacted with antibody at low temperature, washed free of unbound antibody, and warmed at 37 degrees C, cells rapidly cleared their surfaces of AAC. This was not accompanied by formation of FC or endocytosis. In some of these cells, a distribution was observed which suggested a possible centrifugal flow of antigenic sites-perhaps an alternate route for disposal of AAC. None of the drugs tested affected symmetrical redistribution. Repeated attempts at detection and topographical analysis of a tumor-specific antigen on the surface of Rous sarcoma virus-transformed chicken and rat cells have provided no evidence for antibody to such an antigen in the serum of immunized animals. Autochthonous, homologous, and heterologous immunizations of chickens and rats did not produce a detectable antibody response to a virus-specific tumor surface antigen. Preliminary results, however, suggest the expression of an individual-specific (unique) tumor antigen on the surface of Rous sarcoma cells.     

Proliferation, viability, and metabolism of human tumor and normal cells cultured in microcapsule

In this study, we investigated the effect of the microenvironment provided by alginate-poly-l-lysine-alginate (APA) microcapsule with liquefied or gelled core on the proliferation, viability, and metabolism of human cells, including anchorage-dependent MCF-7 breast cancer cells and primary fibroblasts, and anchorage-independent K-562 leukemia cells; cells in conventional culture were used as control. The growth pattern of cells in microcapsule was examined by phase-contrast micrography. The cell viability, proliferation, organization, and gene expression were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, hematoxylin and eosin staining, live/dead staining, 5-bromo-20-deoxyuridine labeling, and immunohistochemistry, respectively. Cell metabolism was determined by measuring glucose and lactate concentrations in medium. The results demonstrate that APA microcapsule with liquefied core provides a microenvironment for both anchorage-dependent and anchorage-independent cells to grow into a large cell aggregate and maintain cell viability at a constant level for a period of time. In conclusion, cells in APA microcapsule are alive and have proliferation potential with lower metabolism rate. APA microcapsule may be a useful tool for in vitro tumor cell modeling and anticancer drug screening as well as for cancer gene therapy. In addition, it lays a solid foundation for the use of microencapsulation in cell culture in vitro and cell implantation in vivo.