Akiyo Shigematsu

Radioluminography for quantitative autoradiography of 14C

SummaryThe aim of this study was to elucidate whether or not quantitative analysis of autoradiographs could be obtained with a [14C]-labelled compound by the use of a new type radiosensor, so called ‘imaging plate’ (IP). Since X-ray films were first used as a radiosensor for macroautoradiography by Ullberg (1), many kinds of X-ray films have been used but the other sensors have never been used. Our results indicated that there was an excellent relation between relative intensity (PSL-BG) and radioactivity on a given area which was completely linear. The linearity was observed in a relatively wide range between 101–105 dpm orders of radioactivity. About 100 times greater sensitivity of the IP than any X-ray film was demonstrated by the use of not only [14C]-radioactive standard sources but also by experimentally provided [14C]-radioactive spots developed on a TLC plate and macroautoradiographs (MARG). In order to obtain reliable quantitative data from a MARG image, it was required that a thin specimen section be kept at a constant level of thickness under freezing condition through out the exposure time.Results also showed that the linear relation of relative intensity versus radioactivity of the specimens had a very wide range from 101–105 dpm/mg of exposure within 7 days. Furthermore, the relation of relative intensity versus relative exposure (radioactivity × exposure time) was linear within the latitude of relative intensity from 101–105 (PSL-BG). Finally, a combination of the IP and BAS2000, computerized image display system, was well equipped to completely erase a background memory before use.

Potassium effect on iron stress in tomato. II. The effects on root CO2-fixation and organic acid formation.

Abstract Tomato plants, two Fe‐efficient cultivars and one Fe‐inefficient cultivar, were grown in treatment combinations of normal and low Fe and K concentrations (Normal Fe, Normal K; Low Fe, Normal K; Low Fe, Low K). Iron‐stressed plants with normal K levels were the first to show elevation of root proton excretion and an enhanced ability to regreen. Roots from the Fe‐efficient cultivars had a marked increase in the rate of CO2 dark fixation that was most prominent with a Normal K, Low Fe. The amount of 14C labeling of root organic acids, particularly malate, citrate, lactate and oxaloacetate increased under Fe‐stress conditions in the Fe‐efficient cultivars; Fe stress with low K resulted in less 14C labeling. A scheme for Fe reduction, uptake and transport is discussed related to CO2 fixation and organic acids (malate, citrate and oxaloacetate.) 1Part of the research done by the senior author while on leave at the Institute for Whole Body Metabolism, Inba, Chiba, Japan. The helpful assistance of Mr. Y....

Pharmacokinetic analysis and antitumor efficacy of MKT-077, a novel antitumor agent

Abstract MKT-077 (1-ethyl-2-{[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-oxothiazolidin-2-ylidenemethyl} pyridinium chloride), a novel rhodacyanine dye in phase I/II clinical trials, may provide a new approach to cancer therapy based on the accumulation in the mitochondria of the cells of certain carcinomas, for example, those of the colon, breast and pancreas. To support the development of MKT-077 for clinical application as an intravenous (i.v.) therapy, we investigated the metabolic fate of [14C]MKT-077 in BDF1 mice as well as the distribution of MKT-077 in experimental LS174T tumor-bearing mice using a high-performance liquid chromatography (HPLC) method. The plasma levels of 14C after i.v. administration of [14C]MKT-077 declined in a triphasic manner. In the first distribution phase, the levels of 14C decreased with a T1/2 of ∼5 min. In the second and terminal phase, the T1/2 of 14C was 2.8–4.6 h and 16.2 h, respectively. Cmax (1 min after injection) increased from 0.3 to 1.5 μg/ml linearly, but less than proportionately between the doses. The AUC(0–∞) at 0.3, 1 and 3 mg/kg were 0.030 ± 0.002, 0.60 ± 0.12 and 1.73 ± 0.25 μg · h/ml, respectively. Plasma clearance was ∼1.8 l/h per kg (at doses of 1 and 3 mg/kg). The steady state volume of distribution (6.8 and 25.1 l/kg) indicated that MKT-077 distributed as a lipid-soluble molecule. The mean residence time (MRT) was 4.1 (at a dose of 1 mg/kg) and 14.1 h (at a dose of 3 mg/kg). In the first rapid phase (5 min after dosing), 14C radioactivity was detected in most of the tissues and organs, most strongly in the kidney cortex, and not in the central nervous system and testes. In the terminal phase (24 h after dosing), 14C contents increased in the intestinal tract, and in the kidney and liver were nearly to the background level. After i.v. bolus administration at a dose of 3 mg/kg of [14C]MKT-077, the predominant route of elimination of the radioactivity was via the feces, and recoveries of total radioactivity in urine and feces corresponded to 33.5% and 61.1%, respectively. More than 60% was recovered within 24 h and 95% within 1 week. MKT-077 was primarily excreted in unmetabolized form with five unidentified metabolites found in the urine and plasma. Intact MKT-077 was retained in the tumor tissue longer than in plasma and kidney in LS174T tumor-bearing mice receiving MKT-077 at an i.v. therapeutic dose (10 mg/kg). This accumulation decreased very slowly, suggesting that the high membrane potentials of tumor cell mitochondria may help retain the drug in tumors.

Stem cell renewal and contraction of the tunica media caused by a damaged blood vessel following a thick needle stab.

SummaryA marked difference in the healing process of the inferior vena cava in rats following a stab with a 17-G (1.48 mmФ)) ultrahard zirconium ceramic (Zr) needle and with a common stainless steel (St) needle (also 1.48 mmФ was observed. This was investigatedin vivo by histological imaging and biochemical micro-autoradiographic imaging using [2-14C]-thymidine as a biomarkerin vivo. On the first day after the stab with either, the Zr or the St injection needle, the tunica adventitia showed the most pronounced damage, as evidenced by a large puncture wound characterized by blood congestion, but with few inflammatory cells being observed. A marked contraction of the tunica media was observed. The depth of the injury reached the tunica layer, but amounted to less than 1/3 of the needle diameter. Loose fragments of the endothelial lining were detected, together with scattered red corpuscles. The survival rate of the experimental animals amounted to less than 40% on the 3rd day after the stab by either the Zr or St needle, due to the large needle diameter. In addition, histological imaging of the wound area in the endothelial layer and tunica media showed considerable congestion and inflammation, which limited the evaluation of the regeneration status of theinferior vena cava of the surviving animals. Results were obtained from a few animals that displayed satisfactory recovery status. On the 3rd day after the stab by either the Zr or St injection needle, a relatively large proportion of the hemostatic clots became incorporated into the collagenous tissue, i.e. the tunica adventitia. A marked contraction of the tunica media was also observed, similar to that on the 1 st day, following the needle injury. In the case of the endothelium (tunica intima), the injury caused by the Zr needle was reinfiltrated by adult stem cells 3 days after the stab, but the tunica media, composed of endothelial cells, still contained relatively contracted collagenous material. In addition, several interesting cell colonies were observed in the medial layer at the short distance from the boundary of the damaged tissue. It was assumed that these colonies produced medial tissue composed of collagenous supporting tissue or smooth muscle cells. In the experiment using the St needle, the incorporation of [2-14C]-thymidine into the nucleus of the stem cells was observed in the small capillaries of the tunica media, but not in the support cells of the latter.

Absorption and translocation of 59Fe and 14C-rhodotorulate in iron-stressed tomato.

Abstract Tomato plants, varieties FER and Earlygirl (both iron efficient), were grown under low Fe conditions for 9 days. Rhodotorulate ‐14C was isolated from Rhodotoruiate pilmanae cultured with 14C‐sucrose. The 14C‐ Rhodotorulate ‐Fe and Rhodotorulate‐ 59Fe were added to the Fe‐stressed plants for 6, 24 and 48 hour periods. It was evident from autoradiograms and tissue sampling that 59Fe and 14C were abundant in roots, stems and leaves. The 14C autoradiograms showed especially high density in the small younger leaves, as was found also with 59Fe. Unlike synthetic chelates rhodotorulate (or metabolized derivatives) were readily absorbed by the roots and translocated to the leaves.

Autoradiographic characterization of newly developed melanoic cell group different from the melanoic tumor piece embedded into a liver lobe of mice

SummarySecondary proliferous melanoic tumors grew at 100% of probability in a liver lobe of C57BL/6 mouse with a piece of B16 melanoma tumor embedded successfully in the lobe. Clear border between host liver tissues and the embedded tumor piece was observed 14 days after embedding. No division image was noted in cells of the embedded tumor piece. In all regions surrounding the embedded tumor piece, there were few accumulations of leucocytes, lymphocytes, or giant cells, indicating a strong likelihood of immune reaction or antigen-antibody reaction, although there was active formation of fibroblast bunds and production of lysosomal enzymes. Secondary proliferous melanoic tumors developed in the environment of liver tissue separately from the embedded tumor location. Results of14C-thymidine- nuclear labeling of embedded tumor cells showed that the majority of the labeled cells did not migrate or divide. In the case of14C-thymidine-labeling of host liver tissue cells, results indicated no transfer of14C-radioactivity into nuclei of embedded tumor cells, although there was positive distribution of14C-radioactivity into nuclei of proliferous cells in the secondary tumor, a newly developed melanoic cell group. When prelabeling was performed the average density of autoradiographic image over the secondary tumor was equivalent to that over the normal liver tissue at the early stage such as 7 days after embedding and decreased with time after that. The newly developed melanoic cells were particularly exciting and proliferous, because they received much more labeling ofl4C-thymidine as compared to liver tissue cells that was provided by one shot labeling of the radioactive tracer 1 h. before sacrifice of tumor embedded mice. There was no developement of secondary melanoic cells in the liver lobe in case of embedding of a tumor piece previously by wrapping with a membranefilter, less 5 μm in pore size.

Radiorespirometric patterns of 14C-substrates in rats. I. Differences in kinds of 14C-substrates and their structural positions labelled with 14C.

SummaryExperiments were conducted by radiorespirometry to elucidate the relation between the respiratoryl4CO2 wave pattern derived from intravenously injectedl4C-substrates and the dispositional partition of these substrates in rats. Two types ofl4C-methionine and 4 types ofl4C-glucose molecules were used as the14C-substrates. The respiratoryl4CO2 wave patterns obtained in experiments using14C-methionine substrates revealed different patterns based on differences in the labelled position and the amount of expired14C-radioactivity as well as the wave height. The experiments usingl4C-glucose substrates reveals that the carbon atoms participate equally in forming the respiratoryl4CO2 wave pattern, regardless of their position in the glucose molecule. Moreover, differences in wave height as a result of differences in position of the labelled carbon atom were not observed. Results of 90-minutel4CO2 radioactivity recovery rate substantiated the explanation of the above-described relationship. The experiments also clarified that wave height is determined by rate of partition to the CO2 pathway of intermediate metabolites arising in the process of labelled14C atom disposition.

The kinetics and dynamics of three kinds of radioactive methionine after i.v. administration in mice

SummaryIn C57BL mice, a tracer amount of methionine (Met) at a concentration approximate to that in the blood was administered in the tail vein (i.V.). The rates of endogenous metabolic decomposition of methionine were obtained by using three specifically labelled compounds, [l-14C]-Met, [m-14C]-Met and [35S]-Met Results on the kinetics and dynamics after i.v. administration suggested that S min after administration, most of the labelled compounds were taken up into the organs and tissues, and only a small portion was excreted in the expired air as CO2, the final metabolite of methionine. At 30 min the radioactive concentration in blood was minimal and was consistent with the maximum of the14CO2 excretion in expired air. Gradual increase of the radioactive concentration in blood after 30 min might be due to the contribution of the metabolite in the blood. Methionine taken up was endogenously utilized by more than 40% during 48 h and it maintained the order of [1-14C] : [m-14C] : [35S].Out of the remaining 60%, 40% was metabolized in 6 h after administration, the [1-14C] moiety being excreted mainly into expired air, the [m-14C] moiety into expired air and urine, and the [35S] moiety mainly into urine and partly into faeces.Microautoradiograms revealed that a part of the last 20% was taken up into the enzyme proteins contained in the pancreatic juice and intestinal juice, and was decomposed within 48 h.

[2-14C]Thymidine incorporation activity of stem cells in either tumor or cradle tissues in a normal or transplanted animals.

Summary A novel autoradiographic procedure was developed for such continuously cycling cells as stem cells on account of proliferating rate of which is astronomically high per min. Negative visualization is observed over any mitotic image by use of a biomarker, “[2- 14 C]thymidine” for a few minutes in both cases, either in vivo or in vitro systems. But, good visualization images were realized by many 14 C-β tracks over stem cells with a few minute labelling of [2- l4 C]thymidine in originated cradles as predicted by Burkitt, H. G(1993). It is clearly elucidated that a short and quick labelling procedure of [2- 14 C]thymidine is useful to evaluate toxicity and efficacy of new drug candidates and to diagnose cluster of unknown malignity or proliferation rate of respective stem cell in in vivo or Ex-vivo system.Results show that the cell proliferation rate of the stem cells in respective tissues was markedly suppressed, dependent on time after dosing and the dose of90Y; 3.7, 37, 370, 3, 700, and 37,000 kBq per mouse (25g). In addition to the above, the sensitivity of the proliferation rate was dependent on amitosis or mitosis and the AUC value of90Y-concentration at specific locations of the cells in the mouse body. The most sensitive cells were the plasmacytoma cells, followed by the pluripotent and unipotent stem cells, the intestinal crypts, epiphysial growth plate and liver cells. Results in this presentation, also gives a clear evidence showing a revival of facultative divider line from G0 stage of epithelium and mascular meditate into the unipotent stem cell cycle. Application of [2-14C]thymidine is useful for evaluation of a grade of maturation in differentiation of malignant cells or replicable unipotent stem cells. development of research in this area.