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Administration of phenylhydrazine to rabbits resulted in the denaturation of hemoglobins in erythrocytes, causing the formation of intracellular precipitates known as Heinz bodies, severe hemolytic anemia, and reticulocytosis. To elucidate the molecular mechanism of the destabilization, we allowed human oxyhemoglobins to react aerobically with phenylhydrazine. After treatment with acetic acid/HCl and H2SO4/methanol, the chloroform extract contained blue-green pigments of major products accompanied by different minor products. Each product was isolated by column chromatography. By fast-atom-bombardment mass spectrometry (FAB-MS) and proton nuclear magnetic resonance (1H-NMR) spectrometry, dimethyl esters of N-phenylprotoporphyrin IX and meso, N-diphenylprotoporphyrin IX were determined. Other major products also were determined to be dimethyl esters of triphenyl-and tetraphenyl-substituted protoporphyrins by FAB-MS. The formation of meso, N-diphenylprotoporphyrin indicated that the addition of a phenyl radical to the meso-carbon atom of the protoporphyrin ring occurred. Triphenyl and tetraphenyl adducts also indicated the formation of phenyl radicals in the aerobic reaction of phenylhydrazine with oxyhemoglobins. From these results, we suggest that the formation of phenyl radicals and the replacement of heme with phenyl-substituted protoporphyrins cause the destabilization of hemoglobins to induce Heinz bodies and hemolytic anemia with phenylhydrazine.

For the purpose to see how the suppression of the nucleic acid synthesis disturbs the cell specialization process the author observed the erythroid cell specialization in anemic rats by treating them with aminopterin (AP) and 5-bromouracil (BU). The observations indicate that the AP injection inhibits the mitosis of erythroblast with the acceleration of hemoglobin synthesis and the denucleation. The bromouracil administration scarcely suppressed the mitosis and the appearance of acidophilicity of erythroblast was retarded. Data indicate that the inhibition of mitosis accelerates the specialization or somatic protein synthesis of erythroblast. The acting mechanisms of the medicaments were discussed from the characteristics of these agents as the analogue of the substances related to DNA metabolism.

With the bone marrow of anemic rats, which had received the repeated injections of phenylhydrazine once a day for three to four days, the effects of aminopterin and bromouracil on the nucleic acid metabolism of erythroblasts were observed in vivo experiment. The injection of aminopterin suppressed DNA synthesis with the lowered labeling index as observed by the incorporation of ³H-thymidine into DNA in vitro. But the grain count per cell showed the level similar to that of anemic control. RNA synthesis was not interfered by AP injections. These results indicate that AP mainly suppresses the thymidilate kinase. Bromouracil showed no such effect even on the administration of a large dose. On the basis of the data obtained from the experiment by using AP, a discussion was made on the correlation between DNA synthesis, nuclear function and the cell specialization.

The disappearance of nucleolus has been traced in the rat erythroid cells in relation with the cell specialization under varying conditions, i. e. in anemia with or without treatment by bromouracil and aminopterin. To make the findings more reliable the observations have been made on tissue section as well as on the smeared samples as the nucleolus becomes often indistinct in smeared cell. The results indicate that under anemic condition nucleolus is lost by the late basoplilic stage. Treatment with bromouracil retained the nucleoli and cytoplasmic basophilicity till later stage of cell specialization suggesting some similar mechanism of RNA disintegration both in nucleolus and cytoplasm.

For the purpose to reveal the correlation between molecular structure and biochemical functions of cytochrome oxidase the author studied purified cytochrome oxidase by using high resolution electron microscope and biochemical methods. 1. Cytochrome oxidase was purified from the cytochrome oxidase-rich submitochondrial membrane (green membrane), obtained from beef heart mitochondria, by three different methods; modification of the method of OKUNUKI et ai., method of FOWLER et ai. and modification of the method ofJACOBS et ai. All the preparations showed a high specific activity under appropriate conditions and consisted mainly of small particles measuring approximately 80 to 90 A. in diameter. 2. The particle, measuring approximately 80 to 90 A. in diameter, took a cylindrical form measuring about 70 A. in diameter at the base and 95 A. in height in an appropriate condition. Many experimental results indicate that the particle is the smallest, fundamental unit of the active cytochrome oxidase. For this reason it was designated as the unit particle of cytochrome oxidase (abbreviated as UPCO). 3. The molecular weight of the unit particle, calculated from its volume and average density (1.24) of lipoproteins (3: 7), was about 270,000. The value was roughly twice the minimum molecular weight of 128, 000 calculated from the heme a content. Accordingly, it is considered that the unit particle contains two heme a molecule and two copper atoms. 4. It was suggested electron microscopically that the particle collected in the 22.6 S position by sucrose gradient ultracentrifugal analysis was a dimer of the unit particle of cytochrome oxidase and also that the particle collected in the 5. 7 S position was a half of the unit particle of cytochrome oxidase. 5. It was also suggested that the particle observed on the green membrane was a subunit of cytochrome oxidase, containing one heme a and one copper atom, and the unit particle of cytochrome oxidase was constituted of two of the particles observed on the green membrane. Namely, the results indicate that the molecular state of cytochrome oxidase on the green membrane apparently differs from that of the purified cytochrome oxidase.

1. A cytochrome oxidase-rich submitochondrial membrane (green membrane) was obtained from beef heart mitochondria after extraction of flavoproteins, cytochrome b, Cll C, etc. by treating with deoxycholate and potassium chloride. 2. The green membrane was formed by self assembly from the membrane fragments (flat sheets), which derived from the cristae membrane of mitochondria and had essentially the same particulate structure as the green membrane. 3. The green membrane exhibited regular arrays of small particles on the surface, measuring approximately 50 to 60 A in diameter with center to center distance of about 70 A. These particles sometime were arranged in a woven structure on the surface. 4. Both the configuration of the particles and the regularity of the arrangement were influenced by detergents and temperature. 5. Green membranes as well as beef heart mitochondria and electron transfer particles commonly retained membrane-structure after sonication and exhibited higher specific activity of cytochrome oxidase than that of purified cytochrome oxidase, if the activity is calculated on the basis of heme a concentration (sec1 / 10 m,lJ.moles of heme a/3 ml). The results suggest that the active sites of cytochrome oxidase are arranged on the surface of these membranes. 6. From these results and other experimental findings, an intimate correlation between cytochrome oxidase and the particles observed on the green membranes is suggested.

For the purpose of settling the specialization stage of erythroblast where the transcription for hemoglobin is initiated, the absorption of heme and the incorporation of tritiated uridine into RNA have been observed on the cells from the anemic rabbit after a mass red cell transfusion by which the DNA synthesis of large size precursors is suppressed and the early denucleation of erythroblasts is stimulated. In the erythroblasts obtained 24 to 72 hours after red cell transfusion a distinct absorption of heme appears first in the proerythroblast, followed by a progressive increase with the advance of the specialization. Hemoglobin synthesis is markedly stimulated after the denucleation. The incorporation of tritiated uridine into RNA is most marked in the proerythroblast and decreases with the advance of specialization stage suggesting that the mRNA synthesis for hemoglobin is initiated at the proerythroblast, continuing to the polychromatic erythroblast where. the synthesis is minimized. The volumetric observations indicate a possible denucleation at proerythroblast, but it has been revealed that the maximum RNA level of macrocytes is comparable to that of early basophilic erythroblast and its highest hemoglobin level is only that expected in the cells denucleated at late basophilic stage. From these observations it has been concluded that the transcription for hemoglobin is triggered at the initial step of erythroid cell specialization, proerythroblast, but it is insufficient for the synthesis of the expected amount of hemoglobin and is compensated or completed by the mRNA synthesis in more advanced stage of specialization.

A previous study has shown that a single injection of ferric nitrilotriacetate (Fe-NTA) produces hepatic parenchymal iron loading in rats. The present paper reports on iron uptake by rat liver and iron toxicity in the liver after a single injection of Fe-NTA (7.5 mg Fe/kg B.W.). Iron uptake was examined with 59Fe-NTA and Fe-[14C]-NTA. Thirty percent of the injected 59Fe was incorporated in the liver non-heme iron fraction at 3 h and retained for 240 h. Only 1% of the 14C injected as Fe-[14C]-NTA was taken up by the liver at 3 h. Gel filtration with a Sephadex G-25 column of the supernatant fraction of the liver obtained 3 h after the injection showed two peaks of 14C activity. One was eluted in the void volume, and the other corresponded to [14C]-NTA. The former had a molecular weight of 5,000-10,000 as determined with a Sephacryl S-300 column and also had 59Fe activity. The electron spin resonance spectra showed that the generation of a free radical in the liver was initiated within 1 h of the iron administration. The free radical generated in the serum by Fe-NTA was revealed to be superoxide by the spin trapping method. These results suggest that Fe-NTA transfers iron to transferrin in the serum and induces hepatic iron loading. Small amounts of the injected iron were taken up by the liver as Fe-NTA and generated superoxide which may have induced lipid peroxidation of the cellular membranes.

The process of hemoglobin sythesis in erythroid cells have been traced mainly by observing cells under the light of 4,060 Å. To scrutinize the theory of hemoglobin synthesis in the nucleus of erythroblasts, several cytochemical and morphological observations were also carried out. The conclusions derived from them are as follows: 1 The absorption at 4,060 Å of the cell, which indicates the location of heme, appeared in the nucleus as early as in the develpmental stage of basophilic erythroblasts. The absorption of hcme in cytoplasm likewise appeared in this stage showing nearly the same intensity of the absorption. The absorption picture of heme in the nucleus, which is coincidental with that of interchromatin, increased along with the progess of maturation as well as in the cytoplasm. The absorption in the nucleus disappeared at the orthochromatic stage where the picture of interchromatin disappeared, while the intensity of absorption in the cytoplasm continued to increase till the stage of reticulocyte. 2 The pseudoperoxidase reaction of hemoglobin, the appearance of acidophlic protein and masked lipids detectable in the location of hemoglobin gave an exactly identical picture with that of the absorption of heme in the nucleus as well as in the cytoplasm. 3 Permeability test performed by supravital staining with Nile blue revealed that the nucleus of erythroblasts from the basophilic to the orthorchromatic stages has increased its permeability being stained selectively as in the case of dead cells. 4 The mitochondria and the endoplasmic reticulum proved to be retained well in the entire course of hemoglobin synthesis, even after the denucleation, the reticulocyte stage. From these observations the authors believe that the hemoglobin syntheis will take place in the cytoplasm throughout the life cycle of erythroid cells, pointing out that the absorption picturebf heme appearing in the nucleus will be in all likelihood due to the infusion of the hemoglobin from the cytoplasm.

Delayed cerebral vasospams is caused by excessive accumulation of dopamine-beta-hydroxylase (DBH) and noradrenaline in cerebral vessel walls. This study demonstrates the mechanisms of delayed spasm, particularly the role of red blood cell components, and the successful relief of delayed cerebral vasospasm. Spasmogenic substances which contained a heme component, such as methemoglobin, methemalbumin and catalase enhanced DBH activity in human serum as measured by a one step chemical spectrophotometric assay. The concentration which gave the highest DBH activity caused the maximum constriction of the basilar artery, when the substances were applied topically. Among components of red cells, methemoglobin, methemalbumin, catalase and nicotinamid adenin dinucleotide (NADH) caused constriction of basilar artery in cats, when applied topically, whereas hematin, hemin and bilirubin caused no significant spasm. An oxyhemoglobin solution obtained by mixture with methemoglobin and ascorbic acid produced no significant vascular spasm either. Relief of delayed cerebral vasospasm was obtained with topical application of specific alpha adrenergic blocking drug such as phenoxybenzamine, specific inhibitors of DBH such as fusaric acid, o-phenanthroline and alphaalpha' dipyridyl beta2 adrenergic stimulants such as salbutamol, and a phosphodiesterase inhibitor, ascorbic acid.