The use of formaldehyde-treated 131-I-albumin in the study of digestive vacuoles and some properties of these particles from mouse liver

The trichloroacetic acid-soluble radioactivity released during incubation of mouse liver particles containing intravenously injected formaldehyde-treated ¹³¹I-albumin consisted almost entirely of ¹³¹I-iodotyrosine. The material was shown to be excreted into the medium and was not due to disruption of the particles by acid. Triton X-100 or the absence of sucrose in the medium inhibited hydrolysis of the particle-associated labeled protein. This inhibition was due to disruption of the digestive vacuoles and dilution of the protein and cathepsins in the suspending medium. These results and other experimental evidence strongly suggest that the ¹³¹I-albumin-containing liver particles are digestive vacuoles. The results also establish that ¹³¹I-albumin may be used to study these vacuoles. High concentrations of sucrose (1 M) inhibited degradation of intraparticulate protein. However, 1 M salts inhibited only the rate of the digestion. Sucrose had an inhibitory effect on a crude cathepsin preparation, and salts stimulated the activity when ¹³¹I-albumin was used as substrate. The effect of high sucrose concentrations as an inhibitor of protein hydrolysis within digestive vacuoles was, therefore, most likely due principally to an inhibition of cathepsin activity within the vacuoles. The effect of salt was probably caused by a stimulation of both intra- and extra-particulate cathepsin activities, although 0.5–1.0 M KCl appeared to protect the particles.     

The development of lysosomal apparatus. II. Incorporation,subcellular distribution,and intraparticulate hydrolysis of 131 I-albumin by liver of mice at perinatal stages

Mouse fetuses at 15th and 18th day of gestation, and newborns aging 0, 5, 10, and 15 days were injected i.p. with 131 I-albumin (RISA). The degree of incorporation by liver and the intraparticulate hydrolysis of RISA in 27,000g × 10 min. particles increased after birth. By this time, changes in subcellular distribution of RISA and marker enzymes were also observed. Following an i.p. injection of India ink, numerous hepatic cells stained with carbon particles were observed at the light microscope from day 5 of life. The results suggest that the lysosomal apparatus acquires full capacity to incorporate colloidal particles and to degrade foreign macromolecules in the first week of life.     

Distribution in tissue homogenates,and the nature of the linkage of injected proteins to subcellular particles

Intravenously injected labeled proteins were recovered mostly in particulate fractions of rat liver homogenate. Distribution showed changes depending on the time elapsed from the injection. ¹³¹I-albumin undergoes an intraparticulate hydrolysis which shows the highest activity in the gradient fractions associated with the highest level of acid phosphatase. The labeled albumin-bearing particles separated at 27,000 g × 10 minutes released their radioactive protein at the same rate as acid phosphatase appeared in the medium, under the effect of such agents as distilled water, salts, homogenization, sonication and pH changes. The substitution of sucrose for distilled water or salts showed that the particles behave as an osmotic system as do lysosomes. These experiments prove that secondary lysosomes involved in the hydrolysis of foreign proteins, whose existence was shown by other authors only at the histochemical level, may survive the distrupting action of conventional homogenization and maintain many properties characteristic of primary lysosomes in addition to the ability of hydrolysing “in vitro” the engulfed material.     

^131I标记Tyr-GX1在荷瘤裸鼠体内分布和显像研究

目的：设计、合成酪氨酸（Tyr）修饰的肿瘤血管靶向肽GX1,研究^131I标记短肽Tyr-GX1在荷人胃癌裸鼠体内的生物学分布与显像,探讨^131I-Tyr-GX1短肽作为肿瘤血管靶向诊治药物的可能性。方法：利用Iodogen碘标法对Tyr-GX1进行131I标记,检测其标记率和体内外稳定性;建立荷人胃癌裸鼠动物模型,尾静脉注射标记肽,分别进行体内生物学分布实验和肿瘤显像实验,结果用PASW Statistics18.0统计软件进行分析。结果：1）.纸层析法结果计算表明,^131I-Tyr-GX1肽的标记率和放化纯均达90%以上;24 h稳定性测试表明,^131I-Tyr-GX1在室温下存放以及与人血清、鼠血清、PBS等溶液混合,其标记率仍然都维持在90%左右,说明其具有良好的体内外稳定性;2）.荷瘤裸鼠体内生物学分布研究显示：标记肽在荷瘤裸鼠双肾放射性计数测量最高;其次是肝脏、肿瘤等组织;脑、骨、肌肉组织放射性计数含量较低,给药24 h时,肿瘤/肌肉（T/M）、肿瘤/血液（T/Bl）、肿瘤/脑组织（T/Br）的放射性比值分别是5.78、4.06和23.01;3）.体内SPECT显像结果显示：尾静脉注射^131I-Tyr-GX1肽后4 h肿瘤部位已开始显影,并随时间的延长,显像逐渐清楚,至18 h时,肿瘤显像最清晰。结论：应用Iodogen碘标法成功标记Tyr-GX1短肽;尾静脉注射^131I-Tyr-GX1后,肿瘤部位可以出现放射性浓聚,表明^131I-Tyr-GX1短肽可以靶向结合于肿瘤部位,有望成为新一种胃肠道肿瘤诊断与治疗的药物。     

STUDIES ON LIVER REGENERATION: I. 131IODODEOXYURIDINE AS A PRECURSOR of DNA IN NORMAL and REGENERATING RAT LIVER

Abstract. ¹³¹Iododeoxyuridine (¹³¹IDU) was injected into normal and partially hepatectomized rats, and the specificity of incorporation of this thymidine analogue into liver DNA was determined 2, 24 and 48 hr following intramuscular injection. At 2 and 24 hr after ¹³¹DU injection, a major proportion of radioactivity in the liver was in the acid-soluble fraction, whereas 48 hr after injection the label in the acid-soluble fraction had decreased considerably. In liver obtained 2 hr after injection of ¹³¹IDU, only 1.8–16.6% of the total radioactivity were in DNA. If, however, the tissue was subjected to formalin fixation, the acid-soluble label was extracted selectively, and of the remaining radioactivity 64–88% was in DNA. Therefore, the radioactivity that is not extracted by formalin may be used as a measure of DNA synthesis at the time of injection of ¹³¹IDU, thus obviating time-consuming biochemical fractionation procedures.     

~(125)Ⅰ—蜂毒肽在小鼠体内分布、吸收、排泄的研究

以氯胺T为氧化剂参照Hunter和Greenwood的多肽类化合物的碘化标记法,制备了~(125)I—标记的蜂毒肽在小鼠体内分布、吸收、排泄的研究,实验证明小鼠肌注~(125)I—蜂毒肽后,吸收很快,主要分布部位为肾、肺、心、肝、小肠、关节、脾与肌肉,脑组织中含量很少,肌肉注射后5分钟血液中含量可达70％,~(125)—I蜂毒肽主要经肾排泄,肌注后30分钟肾脏浓集最高,而尿液中以1.5小时为最高,而粪便中排泄少。     

Heterolysosome formation in mouse liver

When formaldehyde-treated ¹³¹I-albumin was injected into mice, the total liver radioactivity did not change significantly from 5 minutes to 60 minutes after injection. There was a progressive increase with time in the amount of radioactivity associated with liver particles which could be released by osmotic shock; the quantity of material tightly bound to particles, but not releasable by osmotic shock, did not change. At five minutes after injection the liver particles did not release acid-soluble radioactivity into the medium when incubated at 37°. These particles contain the injected protein in osmotically releasable form not associated with proteolytic enzymes and therefore correspond to phagosomes. At 10, 30 or 60 minutes after injection, the particles degraded the protein at similar rates but the activity ceased after 90 minutes incubation when only 50 to 60% of the osmotically releasable material was hydrolyzed. This cessation of activity was shown to be due to a thermal disruption of the particles during incubation.     

The influence of testosterone on the synthesis and degradation rate of various RNA species in the mouse kidney.

1. The effect of a daily injection of testosterone on the in vivo rate of RNA synthesis and degradation in the kidney of castrated mice was determined. 2. Following the administration of testosterone there was a progressive increase in kidney weight, RNA and protein content in the castrated mouse kidney. 3. The rates of synthesis were calculated from the measured incorporation of radioactivity into various RNA species and acid-soluble nucleotides. The kidney RNA was labelled by the injection of a single dose of [5-3H] orotic acid. When the incorporation into ribosomal RNA was at the maximum (48 h), one group of mice was injected with testosterone (100 mug/day/20 g body weight) and the other served as the control. 4. The rates of synthesis and the turnover time were calculated on the basis of the half-life and the rate of decrease of specific radioactivity of RNA. The rates obtained were high in the first 5 days following hormone administration and then slowed down. 5. The results suggest that testosterone changes the rate of synthesis, not the rate of degradation of the mouse kidney RNA. This is consistent with the higher activity of RNA polymerase in testosterone-treated mice as previously observed and described (Avdalovi?, N. and Kochakian, C.D. (1969) Biochim. Biophys. Acta 183, 382-393; Avdalovi?, N. (1970) Biochem. J. 119, 331-338).     

Renal uptake and degradation of trapped-label insulin

In order to study the kinetics of insulin degradation in the kidneys and liver, insulin was labelled by a trapped-label procedure and injected into rats. In contrast to conventional 125I-insulin, the trapped-label preparation allows quantitative measurements of the extent of degradation in vivo because the final degradation products do not leave the cells. One hour after injection, the amount of radioactivity in the kidneys from a trace dose of trapped-label insulin was 10 times higher that from conventionally labelled insulin; over 80% of the increase was due to low molecular weight degradation products which were retained in the kidneys. The amount of acid-precipitable radioactivity in the blood was the same for both labelled preparations, indicating that their rates of clearance were similar. In the kidney, we detected no degradation products of molecular weight intermediate between intact insulin and the end products of proteolysis. After 2 h, 33% of the injected dose remained in the kidneys and only 13% in the liver. Over 80% of the renal radioactivity was sedimentable in an isotonic density gradient, indicating that intact insulin, as well as degradation products in the cells, were enclosed within membrane-bound vesicles.     

Evidence for ethylation of rat liver deoxyribonucleic acid after administration of ethionine

1. Administration of a large dose (500mg/kg body wt.) of (3)H-labelled l-ethionine to rats resulted in the incorporation of a small amount of radioactivity into the liver DNA. Considerable evidence that this radioactivity was not due to contamination of the isolated DNA with labelled protein, RNA, S-adenosyl-l-ethionine or l-ethionine was obtained. 2. After acidic hydrolysis of the DNA isolated from the livers of rats treated with labelled l-ethionine, virtually all of the radioactivity present in the DNA was found in a fraction with similar chromatographic properties to 7-ethylguanine. 3. Treatment of rats with comparable doses of l-methionine did not lead to the formation of 7-methylguanine in the liver DNA. 4. These results are discussed in relation to the induction of liver tumours by ethionine.     

Kinetics of indium-III labelled lymphocytes in normal subjects and patients with Hodgkin's disease.

The distribution in the body and the circulation in the blood of autologous lymphocytes labelled with indium-III were studied in two normal subjects and two patients with Hodgkin''s disease. Four hours after injection radioactivity was identified in the spleen, liver, and bone marrow. Radioactivity, followed by imaging and whole body scanning, began to appear in the lymph nodes four to 18 hours after injection, and some, though not all, lymph node groups in the body could be readily visualised. There were no differences between the normal subjects and the patients with Hodgkin''s disease. The pattern of clearance of radioactivity from the blood was consistent with a normal circulation between blood and lymphoid tissues of the labelled lymphocytes. Since indium-111 stays firmly attached to the cell, it seems an ideal label for studying lymphocyte kinetics, and the use of this technique may have further clinical application.     

Regulation of mammalian protein synthesis in vivo. Protein synthesis in rat liver and kidney after the administration of sublethal doses of cyclohyximide.

Protein synthesis in vivo was studied at various times after the administration of sublethal doses of cycloheximide to rats. Cycloheximide caused an inhibition, followed by a dose-and time-dependent stimulation, of incorportation of labelled precursor into proteins of the liver and kidney. The stimulation of protein synthesis at 24h was not due to a change of precursor pool or the specific radioactivity of the precursor used. During the stimulatory period, leucine incorporation into various cellular protein fractions varied; incorporation into total nuclear protein was the most affected.     

Incorporation of orotic acid into nucleotides and RNA in mouse organs during 60 minutes.

Mouse kidney and liver were found to increase their levels of radioactivity above that of serum from 2 to 60 min after administration of [6-14C]orotic acid. In spleen, thymus and brain, the radioactivity level reached a maximum soon after the injection and then decreased, as did that in serum. Sixty minutes after the injection, 44% of the administered isotope dose was found in the kidneys, 22% in the liver and 0.75% in the spleen. The 14C activity in liver UTP increased rapidly and then remained constant for 60 min. The ratio between the activities in uridine phosphates and UDP-sugars was 3:4 from 10- 60 min after injection. In the liver and kidneys, the RNA 14C activities at 60 min after injection were 15% of the activity in their acid-soluble fractions. Intraperitoneal administration was found to be preferable to intravenous administration for studies on nucleotides and RNA in mouse liver, due to the delayed incorporation of the [14C]orotic acid activity into the nucleotide pool.     

STUDIES ON LIVER REGENERATION: II. EFFECTS of PHENOBARBITAL ON the ONSET and PATTERN of RAT LIVER REGENERATION FOLLOWING PARTIAL HEPATECTOMY

Abstract. Partially hepatectomized rats were given a single intraperitoneal injection of a phenobarbital solution or of water immediately after surgery. At various time intervals following the operation, the animals were injected with ¹³¹ iododeoxyuridine (¹¹³IDU), sacrificed 2 hr later, and radioactivity retained in formalin-fixed liver tissue was determined as a measure of DNA synthesis at the time of administration of the labeled precursor. In control animals without phenobarbital treatment, ¹³¹IDU incorporation into liver began to increase between 14 and 16 hr after partial hepatectomy. Phenobarbital treatment (0.1 mg per g of body weight) resulted in a delay of the increase in ¹³¹IDU incorporation by several hours. This delay was observed in animals subjected to partial hepatectomy in the morning as well as in those operated on in the evening. After phenobarbital treatment, the increase of mitotic activity was either delayed or occurred more slowly. the results are compared with the reported effects of partial hepatectomy on the time course of microsomal enzyme induction by phenobarbital.     

Metabolism of liposomes prepared from a labelled ether analog of 1,2-dioleoyl-sn-glycero-3-phosphocholine in the rat

To synthesize the ether analog of 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1-O-cis-9'- octadecenyl -2-O-cis-9'-[9',10'(n)-3H] ocatadecenyl -sn-glycero-3- phosphocholine, we have adapted available methodology and have obtained a product of high specific activity and purity. The labelled dioleyl ether phosphatidylcholine ( DOEPC ) was used to prepare 250-350 A unilamellar liposomes, which contained also PC and free cholesterol. Following intravenous injection into rats, labelled PC was cleared from the plasma at a faster rate than DOEPC . The uptake of both labelled compounds by the liver increased up to 3 h, at which time there was about 40% of injected PC and 60% of DOEPC . The PC disappeared more rapidly than the DOEPC , so that 17 and 48% of injected label were present in the liver 24 h after injection of PC and DOEPC , respectively. Ten days after injection of DOEPC , about 10% of the label was still present in the liver. During the first 5 days after injection of DOEPC , 10% of radioactivity was found in the gastrointestinal tract and about 20% in the carcass; no increase in carcass radioactivity occurred during the loss of label from the liver. 24 and 48 h after injection of DOEPC , 40% of liver radioactivity was present in a neutral lipid, which on TLC comigrated with triacylglycerol. Since after alkaline hydrolysis this compound comigrated with diacylglycerol, it appears that the ether bond of DOEPC was not hydrolyzed, but after removal of phosphocholine, presumably by phospholipase C, the diether glycerol was reacylated . In experiments in vitro, the rate of exchange of labelled PC with red blood cell phospholipids exceeded that of DOEPC . Incubation of cultured hepatocytes with liposomes containing PC and/or DOEPC resulted in uptake of both phospholipids and metabolism of DOEPC to neutral lipids. The present findings indicate that DOEPC undergoes slow metabolism and can be eliminated from the body. These properties could prove advantageous for the use of DOEPC as a carrier of drugs and possibly as a carrier of free cholesterol in reverse cholesterol transport.     

Biosynthesis of heterocyst glycolipids of Anabaena cylindrica

The incorporation of sodium acetate-[1-¹⁴C] into the heterocyst glycolipids of Anabaena cylindrica cultures from 60–234 hr old is reported. Incorporation of radioactivity was maximal in 88 hr old cultures. In 60 hr and 88 hr cultures about 90 % of the radioactivity of the heterocyst glycolipids was found in the non-saponifiable glycolipid fraction, whereas in older cultures this fraction contained only 75 % of the radioactivity. Acid hydrolysis of non-saponifiable heterocyst glycolipid fractions showed that in 60 hr cultures, 81 % of the radioactivity occurs in the lipid moiety, whereas in older cultures a greater proportion (40–53 %) of the radioactivity was found in the sugar residue. The lipid fraction obtained by acid hydrolysis contained a mixture of labelled long chain mono-, di- and trihydric alcohols. In young (60 hr) cultures the primary alcohol fraction was most heavily labelled (57.3 % of the radioactivity in the non-saponifiable glycosides) with much smaller amounts in the diol and triol (8.4 and 15.1 % respectively), whereas in older cultures (234 hr) the primary alcohol (23.6 %) diol (22.5 %) and triol (18.9 %) fractions contained ca equal amounts of radioactivity.     

Uptake of iodinated human kidney alpha-D-mannosidase by rat liver- Association with membrane elements and stability in vivo and in vitro.

1. Human kidney alpha-D-mannosidase (form A) was labelled with 125I to a specific radio-activity of approx. 2250muCi/mg of protein, essentially without loss of enzymic activity. The enzymic activity and radioactivity of the iodinated material also co-migrated in gel filtration and gel electrophoresis. 2. The binding of 125I-labelled mannosidase in vitro to particulate material in liver and kidney homogenates was of the other of 2 pg/mg of particulate material in liver and kidney homogenates was of the order of 2pg/mg of particulate protein withing 16h at 37 degrees C, and essentially zero in intervals of up to 60 min. The degradation in vitro of labelled exogenous mannosidase was of the order of 10-20pg/ 16th per mg of protein in postnuclear supernatant, and it was saturated entirely within 1h at 37 degrees C. 3. The binding of labelled mannosidase in vivo to particulate elements of liver homogenates 60 min after intravenous injection was at least 10 times higher in terms of specific radioactivity than the highest value attainable in vitro. Virtually all exogenous enzyme bound to liver particulate material could be recovered in macromolecular form after disruption of membranes by detergents. 4. The radioactive enzyme bound to liver particulate material could be detached almost completely by shearing, repeated freezing and thawing, and exposure to strong detergents under conditions that do not eliminate rough-endoplasmic-membrane structure. It could bot be released, however, by high salt concentration (0.5M-KC1) or by exposure to weak detergents such as Tween 80. The particle-bound enzyme should thus be associated with plasma membranes and lysosome-like elements. 5. Of the rat tissues studied, only liver could approach, within 60 min after the injection, the concentration of exogenous mannosidase found in the blood serum. The activity per g tissue weight fell progressively from liver (60% of serum value) to kidney (16% of serum value), lung (8% of serum vlaue), spleen (6% of serum value) and brain (0.9% of serum value). Most of the radioactive enzyme found in tissues other than liver appeared to be present in a free form, whereas in liver more than 50% of the labelled enzyme was associated with membrane elements.     

Methylation of nuclear proteins by dimethylnitrosamine and by methionine in the rat in vivo

1. The incorporation of methyl groups into histones from dimethylnitrosamine and from methionine was studied by injection of the labelled compounds, isolation of rat liver and kidney histones, and analysis of hydrolysates by column chromatography. 2. Labelled methionine gave rise to labelled in-N-methyl-lysine, di-in-N-methyl-lysine and an amino acid presumed to be omega-N-methyl-arginine. 3. Administration of labelled dimethylnitrosamine gave rise to labelled S-methylcysteine, 1-methylhistidine, 3-methylhistidine and in-N-methyl-lysine derived from the alkylating metabolite of dimethylnitrosamine. In addition, labelled formaldehyde released by metabolism of dimethylnitrosamine leads to the formation of labelled S-adenosylmethionine, and hence to labelling of in-N-methyl-lysine, di-in-N-methyl-lysine and omega-N-methylarginine by enzymic methylation. 4. The formation of in-N-methyl-lysine by alkylation of liver histones was confirmed by using doubly labelled dimethylnitrosamine to discriminate between direct chemical alkylation and enzymic methylation via S-adenosylmethionine. These experiments also suggested the possibility that methionine residues in the histones were alkylated to give methylmethionine sulphonium residues. 5. The extent of alkylation of liver histones was maximal at about 5h after dosing and declined between 5 and 24h. The methylated amino acids resulting from direct chemical alkylation were preferentially lost: this is ascribed to necrosis of the more highly alkylated cells. 6. Liver histones were about four times as alkylated as kidney histones; the extent of alkylation of liver histones was similar to that of liver total nuclear proteins. 7. Methyl methanesulphonate (120mg/kg) alkylated liver histones to a greater extent than did dimethylnitrosamine. Diethylnitrosamine also alkylated liver histones. 8. The results are discussed with regard to the possible effects of alkylation on histone function, and the possible role of histone alkylation in carcinogenesis by the three compounds.     

Anabolism versus catabolism of [5-3H]uridine and its relationship to ribonucleic acid labelling in mouse liver after partial hepatectomy

The balance between anabolism and catabolism of [5-(3)H]uridine was studied in the mouse after partial hepatectomy. Labelling of RNA and UDP-glucose was determined and evaluated in relation to changes in the specific radioactivity of UTP. The amounts of labelled catabolic products of uridine were increased several-fold in liver and blood after partial hepatectomy. The specific radioactivity of RNA decreased to about 60% of the control value at 6h and was in the same range as that of control liver at 24h after operation. Decreased labelling of RNA and UDP-glucose was attributable to decreased specific radioactivity of UTP. No changes in the size of the UTP pool or in the balance between uridine anabolism and catabolism were found that could explain the decreased specific radioactivity of UTP. Rather, the alterations in the labelling of this metabolite induced by the partial hepatectomy may be related to decreased phosphorylating capacity in the liver cells and/or dilution of the labelled precursor in an expanded uridine pool. The enhanced amounts of uridine catabolic products in liver and blood were probably a consequence of accumulation and altered incorporation of the metabolites from the blood into the liver cells. Despite the increased amounts of labelled catabolic products and the decreased labelling of RNA, the results reported here actually suggest decreased uridine catabolism and slightly increased RNA synthesis in mouse liver after partial hepatectomy. The results stress the importance of proper controls in determination of nucleic acid synthesis and in metabolic studies by use of labelled precursors.     

Inosine metabolism in the rat

1. Uptake and subsequent metabolism of purine and ribose moieties was monitored after intravenous administration of doubly labelled inosine. 2. More than 95% was cleared from the plasma within 5 min, and 99% within 20 min. 3. Approx. 50% of the 160 mumol total was rapidly incorporated into liver and kidney. Kidney removed the greatest amount (21 mumol/g wet wt.), about 10-fold more than heart, lung or liver. Lung and heart accounted for only 3%. These tissues then lost radioactivity during the remainder of the experiment. Radioactivity in the skeletal muscle, in contrast, increased from 8% of the injected dose at 5 min to 40% at 60 min. 4. In liver, kidney, heart and lung there was a significant difference in the fate of inosine. After initial incorporation of inosine, kidney predominantly lost inosine; heart preferentially lost purines; lung preferentially lost ribose radioactivity; and in liver the ribose radioactivity was rapidly lost, whereas purine was retained. Some of the ribose moiety was metabolized to glucose, presumably in the liver, and then released into the blood. Ribose radioactivity (probably as glucose) and radioactive hypoxanthine accumulated in skeletal muscle throughout the experiment. 5. Inosine caused a rapid and prolonged increase in the blood glucose content, from 6 to 15 mM in 60 min. This was accompanied by a small increase in plasma insulin. 6. It is concluded that the purine and ribose radioactivity lost from the kidney, liver and other tissues becomes incorporated into skeletal muscle.