Diacytosis of human asialotransferrin type 3 in the rat liver is due to the sequential engagement of two receptors

The possible role of transferrin receptors in the diacytosis of human asialotransferrin type 3 (HAsTf-3) by the rat liver was studied in vivo. A trace dose of the ligand was allowed to compete for hepatic binding sites against diferric transferrin, the concentration of which was varied between 5 400- and 18 000-fold. Binding of HAsTf-3 was insensitive to the presence of 2Fe-transferrin in this range, and the liver bound the ligand equally efficiently, regardless of whether it was presented in the holo or apo form. In contrast, pretreating the animals with desialylated bovine submaxillary mucin (2 mg/100 g, 2 min before the dose) prevented the asialotransferrin-liver interaction. These findings indicate that endocytosis of HAsTf-3 is mediated by the Gal/GalNAc-specific lectin and not by transferrin receptors. Although 2Fe-transferrin did not affect binding, it did reduce the half-life of the ligand in the liver, thus suggesting that transferrin receptors play an important role in the exocytic leg of the diacytic cycle. Based on our present and earlier data, a model is proposed in which the engagement of lectin and transferrin receptor in the diacytic cycle is envisaged sequentially so that HAsTf-3 switches receptors at an acidified subcellular site.     

Galactose-specific elimination of human asialotransferrin by the bone marrow in the rabbit

Rabbit bone marrow accretes and degrades human asialotransferrin in vivo through a mechanism that recognizes the exposed galactose groups in this glycoprotein. After the liver, rabbit bone marrow is the second mammalian tissue now being identified as possessing a galactose-specific pathway for the elimination of a plasma protein. However, comparative studies with desialylated glycoproteins containing bi-, tri-, and tetraantennary glycans (asialofetuin, asialoorosomucoid, chicken α₁-acid glycoprotein, and rabbit transferrin) indicate that the bone marrow recognizes fewer glycan structures than does the liver. Optimal uptake and degradation of an asialoglycoprotein by the bone marrow requires the presence of biantennary glycans.     

The effects of a number of non-steroidal anti-inflammatory compounds on parturition in the rat

A method was developed for assessing non-steroidal anti-inflammatory compounds for their potency in blocking parturition, and prolonging gestation, in the rat. This consisted of injecting compounds into groups of 10 to 13 rat dams twice daily from Day 18 through Day 22 of pregnancy, and comparing the treated dams with appropriate controls on Day 23. The rate of blocked parturition appeared to be positively related to dose of non-steroidal anti-inflammatory agent and, therefore, this model and end-point appeared to be useful for assessing different non-steroidal anti-inflammatory compounds for potency. Among the twenty-seven non-steroidal anti-inflammatory agents evaluated by this method were: ibuprofen, phenylbutazone, tolmetin, flufenamic acid, 2(p-biphenyl) acetic acid, mefenamic acid, aspirin, fenoprofen calcium, flumazole, ketoprofen, naproxen, isoxicam, indomethacin, 2(p-biphenyl) propionic acid, 2(2′-fluoro-4-biphenyl) propionic acid, flurbiprofen, sudoxicam and piroxicam. Piroxicam, sudoxicam, flurbiprofen, 2(p-biphenyl) propionic acid and 2(2′-fluoro-4-biphenyl) propionic acid showed the greatest potency. The relationship between structure and activity and between the blocking of parturition and the inhibition of prostaglandin synthesis are discussed.     

The effects of standard neuroleptic compounds on the binding of 3H-spiroperidol in the striatum and mesolimbic system of the rat in vitro.

Neuroleptics are reported to produce their antipsychotic activity and extrapyramidal side effects by blocking dopamine receptors in the mesolimbic system and striatum respectively. We have thus looked at the characteristics of the binding of ³H-spiroperidol to specific binding sites in these two areas of rat brain and the ability of a number of neuroleptics to displace it from these sites.The ³H-spiroperidol binding sites in the striatum and mesolimbic area are different and evidence has been obtained for an involvement of 5-HT receptors, particularly in the latter area.In the striatum the order of activity of neuroleptics in displacing ³H-spiroperidol binding parallels their clinical potency. This is not the case in the mesolimbic system. Also the ratio of activity of a neuroleptic in the two brain areas does not correlate with its ability to produce extrapyramidal disturbance in man. This may be due to the interaction of neuroleptics, particularly in the mesolimbic system, with receptors not involved in the expression of antipsychotic activity.     

Structure--function studies of oligosaccharides of recombinant human thyrotrophin by sequential deglycosylation and resialylation

Recombinant human thyrotrophin (rhTSH) contains oligosaccharidesterminating in -galactose-sialic acid, and had lower metabolicclearance and higher in vivo bioactivity compared to pituitaryhTSH, which has oligosaccharides terminating predominantly in-N-acetylgalactosamine-sulphate. Previous studies using completeremoval of the oligosaccharide chains showed an important rolefor the carbohydrate in the biological activity of the hormone.In the present study, we have determined the contribution ofthe individual monosaccharides to hormonal activity by sequentialdeglycosylation of rhTSH using exoglycosidases. We have alsoinvestigated the effect of resialylation of desialylated rhTSHusing sialyltransferases. Sequential removal of sialic acid,galactose or N-acetylglucosamine resulted in a > 10-foldincrease in the in vitro bioactivity of rhTSH. The metabolicclearance of the derivatives was faster than that of intacthormone, but agalacto-rhTSH was cleared slower than asialo-rhTSH.However, the in vivo bioactivity decreased progressively witheach monosaccharide removal. The increased cyclic AMP-stimulatingactivity, increased metabolic clearance and the decreased invivo biologic activity were all reversed by resialylation ofthe terminal galactose residues. These results indicate thatthe in vitro, as well as the in vivo, bioactivities of rhTSHare modulated by terminal sialylation. The effects of sequentialdeglycosylation on the in vitro activity of rhTSH are differentfrom those reported earlier for human chorionic gonadotrophin.Thus, modification of the oligosaccharides by glycosidases andglycosyltransferases can be used as a powerful tool to delineatethe function of carbohydrate in glycoproteins and to engineermore potent hormone analogues with a longer half-life and/orhigher bioactivity. deglycosylation exoglycosidases oligosaccharides recombinant thyrotrophin sialyltransferases     

Studies of the metabolism of asialotransferrins: the metabolic heterogeneity of human asialotransferrin.

Catabolism of human transferrin and human asialotransferrin was simultaneously studied in guinea pigs by means of total body radioactivity measurements. Total body activity representing transferrin decreased at a constant rate with an average half-life of 88 h. Decrease of the total body activity representing asialotransferrin exhibited at least two rates; the half-life of the fast initial component averaged at 25 h, whereas the half-life of the slower component averaged at 55 h. Transition occurred between the 50th and 80th hours of the experiments. The complex character of the elimination curves could not be explained by differences in the iron content of asialotransferrin, by the presence of transferrin variants or of denatured protein in the injected material, by residual sialic acid in the preparations, by accumulation of radioactive terminal catabolic products in the body, by an association of asialotransferrin with any other macromolecular plasma constituent, by changing conditions for mass action, or by a continuing return of labeled protein from the extravascular space. Injection of bovine asialotransferrin into guinea pigs did not result in complex total body curves. Analyses of guinea pig tissues demonstrated that human asialotransferrin had marked affinity for the liver and none for the kidney, lung, or spleen. These observations are consistent with the hypothesis that the glycopeptides in human transferrin are heterogeneous in that removal of the sialyl residues exposes structures with different affinities for the hepatic asialoglycoprotein receptor. The precise chemical basis for the metabolic heterogeneity is unknown.     

The effects of vanadium compounds on the activation of adenylate cyclase from rat adrenal membrane

In rat adrenal membrane, vanadyl sulfate, but not vanadate, inhibits the nonhydrolyzable GTP analogs-, forskolin- and NaF-stimulated activation process of adenylate cyclase. In these reactions, the half-maximum concentration of vanadyl for inhibition was approx. 0.3 mM. The binding of [3H]guanyl-5'-yl imidodiphosphate to the membrane (Kd = 2 microM) was not affected by vanadyl sulfate under the conditions in which the vanadyl sulfate inhibits the activation process. Also, the binding of ACTH to its receptor was inhibited by neither vanadyl sulfate nor vanadate, and the catalytic unit of adenylate cyclase appears to be unaffected by vanadyl sulfate. When the activation by nonhydrolyzable GTP analog was enhanced by Ca2+, vanadyl sulfate strongly inhibited the activation of adenylate cyclase.     

The fate of beryllium compounds in the rat

Beryllium was injected intravenously into rats. When administered as ionic solution or as citrate complex, nearly half of the injected quantity was excreted and most of the remaining beryllium was retained in the bones.     

Studies of the metabolism of asialotransferrins: nonspecific changes in the metabolic behaviour of human asialotransferrin in avians.

In a previous study (Regoeczi, E. & Hatton, M.W.C. (1976) Can J. Physiol. Pharmacol. 54, 27-34) it was shown that the chicken (Gallus domesticus) does not possess a hepatic asialoglycoprotein receptor. In the present study the same conclusion is drawn for the duck (Anas platyrhynchos). For this reason these avian species were used to assess those changes in the distribution and catabolism of human asialotransferrin which takes place in the absence of the asialoglycoprotein receptor of the liver.     

Taste effects of some amino acids and glutamate compounds in the rat

Behavioral responses to five L-amino acids (Gly, Arg, Leu, Ala,Met) and five related L-glutamate compounds (MSG, MKG, MAG,Gln, GluHCl) were measured using 1-min taste reactivity andstandard 24-h, two-bottle preference tests. Taste reactivitytests measure the immediate pattern of ingestive and aversiveoral motor behavior elicited by direct oral infusion of tastestimuli. By permitting acute observations in non-deprived rats,taste reactivity tests are more sensitive to taste factors thanstandard long-term tests. Three stimulus concentrations of eachcompound were selected by behavioral and electrophysiologicalcriteria. Taste reactivity results often conflicted with standardintake results. In taste reactivity tests both Gly and MSG elicitingestive oral motor responses that increase with stimulus concentrationin the absence of aversive behavior. The opposite responseswere obtained using long-term intake tests; MSG and Gly preferenceratios actually decrease with increasing concentration. Thesedata suggest a reinterpretation of standard, longterm intaketests. Specifically, effects of taste versus post-oral stimulimay be distinguished by contrasting taste reactivity and two-bottlepreference tests. Differences in the pattern of oral motor behaviorselicited by the amino acid and glutamate compounds are alsodiscussed.     

Intracellular movement of cell surface receptors after endocytosis: resialylation of asialo-transferrin receptor in human erythroleukemia cells

The intracellular movement of cell surface transferrin receptor (TfR) after internalization was studied in K562 cultured human erythroleukemia cells. The sialic acid residues of the TfR glycoprotein were used to monitor transport to the Golgi complex, the site of sialyltransferases. Surface-labeled cells were treated with neuraminidase, and readdition of sialic acid residues, monitored by isoelectric focusing of immunoprecipitated TfR, was used to assess the movement of receptor to sialyltransferase-containing compartments. Asialo-TfR was resialylated by the cells with a half-time of 2-3 h. Resialylation occurred in an intracellular organelle, since it was inhibited by treatments that allow internalization of surface components but block transfer out of the endosomal compartment. Moreover, roughly half of the resialylated molecules were cleaved when cells were retreated with neuraminidase after culturing, indicating that this fraction of the molecules had returned to the cell surface. These results suggest that TfR is transported from the cell surface to the Golgi complex, the intracellular site of sialyltransferases, and then returns to the cell surface. This pathway, which has not been previously described for a cell surface receptor, may be different from the route followed by TfR in iron uptake, since reported rates of transferrin uptake and release are significantly more rapid than the resialylation of asialo-TfR.     

岩藻多糖在2型糖尿病大鼠模型的作用及机制研究

目的:研究岩藻多糖(Fucoidan)对链脲佐菌素诱导实验性2型糖尿病大鼠的治疗作用.方法:在SD大鼠建立2型糖尿病动物模型,随机分为5组,分别用生理盐水、岩藻多糖高、中、低剂量(600mg/kg、400mg/kg、150mg/kg)、二甲双胍(200mg/kg)灌胃给药30天,同时采用正常SD大鼠为正常对照.测定各组大鼠体重、空腹血糖、血脂和血清胰岛素水平.结果:高、中剂量岩藻多糖能够明显降低模型动物空腹血糖水平,降低空腹胰岛素水平,改善胰岛素抵抗,降低血清胆固醇、甘油三酯、低密度脂蛋白胆固醇含量(P<0.05),升高高密度脂蛋白胆固醇含量(P<0.05),其作用效果与阳性对照药二甲双胍效果相当.低剂量岩藻多糖作用效果不明显.结论:岩藻多糖有降血糖、调节血脂紊乱的功能,时实验性2型糖尿病大鼠有治疗作用.     

The interaction in vivo of transferrin and asialotransferrin with liver cells.

Rat transferrin or asialotransferrin doubly radiolabelled with 59Fe and 125I was injected into rats. A determination of extrahepatic and hepatic uptake indicated that asialotransferrin delivers a higher fraction of the injected 59Fe to the liver than does transferrin. In order to determine in vivo the intrahepatic recognition sites for transferrin and asialotransferrin, the liver was subfractionated into parenchymal, endothelial and Kupffer cells by a low-temperature cell isolation procedure. High-affinity recognition of transferrin (competed for by an excess of unlabelled transferrin) is exerted by parenchymal cells as well as endothelial and Kupffer cells with a 10-fold higher association (expressed per mg of cell protein) to the latter cell types. In all three cell types iron delivery occurs, as concluded from the increase in cellular 59Fe/125I ratio at prolonged circulation times of transferrin. It can be calculated that parenchymal cells are responsible for 50-60% of the interaction of transferrin with the liver, 20-30% is associated with endothelial cells and about 20% with Kupffer cells. For asialotransferrin a higher fraction of the injected dose becomes associated with parenchymal cells as well as with endothelial and Kupffer cells. Competition experiments in vivo with various sugars indicated that the increased interaction of asialotransferrin with parenchymal cells is specifically inhibited by N-acetylgalactosamine whereas mannan specifically inhibits the increased interaction of asialotransferrin with endothelial and Kupffer cells. Recognition of asialotransferrin by galactose receptors from parenchymal cells or mannose receptors from endothelial and Kupffer cells is coupled to active 59Fe delivery to the cells. It is concluded that, as well as parenchymal cells, liver endothelial and Kupffer cells are also quantitatively important intrahepatic sites for transferrin and asialotransferrin metabolism, an interaction exerted by multiple recognition sites on the various cell types.     

The different effects of recombinant human tumor necrosis factor on rat fibrosarcoma sublines

Summary The antitumor effect of recombinant human tumor necrosis factor (rH-TNF) on two clones of rat fibrosarcoma with different metastatic potential to lymph nodes was examined. The colony formation of clone A, which has high metastatic potential, was completely inhibited by continuous exposure to rH-TNF at 50 U/ml. In contrast, colony formation of clone G, which has low metastatic potential, was not inhibited by high concentrations of rH-TNF (10,000 U/ml). The inhibitory effect of rH-TNF on colony formation by clone A was also observed with a 1-h exposure to rH-TNF. This effect was time and concentration dependent, as determined by the colony assay, ³H-thymidine uptake assay, and ⁵¹Cr-release assay. ³H-thymidine and ³H-uridine uptake per cell of clone A exposed to rH-TNF was not decreased. This suggests that the mechanisms of the antitumor effect of rH-TNF were not due to inhibition of DNA and RNA synthesis of tumor cells. In vivo growth and lymph node metastases of clone A inoculated i.p. to Donryu strain rats were completely suppressed by 14 consecutive i.p. injections of 10⁵ or 10⁶ U/kg per day of rH-TNF. On the other hand the growth of clone G was not influenced by rH-TNF administration.