Two forms of erythropoietin, EPO-bi and EPO-tetra, with different biological activities were isolated from the culture medium of a recombinant Chinese hamster ovary cell line, B8-300, into which the human erythropoietin gene had been introduced. EPO-bi, an unusual form, showed only one-seventh the in vivo activity and 3 times higher in vitro activity of the previously described recombinant human EPO (standard EPO). In contrast, EPO-tetra showed both in vivo and in vitro activities comparable to those of the standard EPO. EPO-bi, EPO-tetra, and the standard EPO had the same amino acid composition and immunoreactivity. However, structural analyses of their N-linked sugar chains revealed that EPO-bi contains the biantennary complex type as the major sugar chain, while EPO-tetra and the standard EPO contain the tetraantennary complex type as the major sugar chain. From examination of various preparations of recombinant human EPO, we found a positive correlation between the in vivo activity of EPO and the ratio of tetraantennary to biantennary oligosaccharides. These results suggest that higher branching of the N-linked sugar chains is essential for effective expression of in vivo biological activity of EPO.Human erythropoietin (EPO) is a glycoprotein hormone that plays a major role in regulating the level of circulating erythrocytes (1) by stimulating the maturation of late erythroid progenitor cells into proerythroblasts (2). In the normal human adult, EPO is produced in the kidney (3). EPO was first purified in a small amount from urine of aplastic anemia patients (4). Several recombinant human EPOs produced in mammalian (5-9) and nonmammalian cells (10) have recently become available, but their biological activities differ from cell to cell. Since the structures of their polypeptide moieties are the same, such variation in activity was suspected as being due to the differences in their carbohydrate moieties. The sugar chain structures of urinary human EPO and recombinant human EPO produced in Chinese hamster ovary (CHO) cells were determined independently by us (11) and by Sasaki et al. (12). Both urinary and recombinant human EPOs contain -40% carbohydrate in the form of three N-linked and one O-linked oligosaccharide chain. Important roles of the carbohydrate moiety in the solubility (13, 14), biosynthesis (14), and biological activity of EPO (9, 15) have been reported. Desialylation of EPO caused complete loss of its hormonal activity in vivo (15, 16) as the asialo-EPO was trapped in the liver (17) by the hepatic asialoglycoprotein binding protein (18) and was rapidly cleared from the circulation. Galactose oxidase treatment of asialo-EPO restored part of the biological activity (16). These results suggested that EPO possesses full biological activity only when it is sufficiently sialylated to avoid clearance by the hepatic asialoglycoprotein binding protein. During the course of study of the productivity of EPO in several recombinant CHO cell lines, we found a unique cell line, B8-300, which produced ...
Antibacterial proteins are important participants in the innate immunity system. Elafin and SLPI are the whey acidic protein (WAP) motif proteins with both antibacterial activity and antiprotease activity, and their role in innate immunity is under intense investigation. We cloned two novel antibacterial WAP motif proteins from mice, SWAM1 and SWAM2. SWAM1 and SWAM2 are composed of a signal sequence and a single WAP motif that has high homologies with the WAP motifs of elafin and SLPI. SWAM1 is constitutively expressed in kidney and epididymis, and is induced in the pneumonic lung. SWAM2 is constitutively expressed in tongue. SWAM1 and SWAM2 inhibit the growth of both Escherichia coli and Staphylococcus aureus at a IC90 (concentration that achieves 90% inhibition) of 10 μM. Human genes LOC149709 and huWAP2 are considered to be human SWAM1 and SWAM2, respectively. These and several WAP motif proteins (WAP1, elafin, SLPI, HE4, eppin, C20orf170, LOC164237, and WFDC3) form a gene cluster on human chromosome 20, suggesting that they may be derived from the same ancestral gene by gene duplication. Our results underscore the role of the WAP motif as a skeletal motif to form antibacterial proteins, and warrant the study of antibacterial activity in other WAP motif proteins.
Influences of galactosylsucrose and xylosylfructoside on body mass gain, the digestive tract mass and concentrations of organic acids such as acetic, propionic, butyric, lactic and succinic acid in the cecum were compared among rats fed a cholesterol-enriched fiber-free diet [cholesterol 6 + cholic acid 1.5 (g/kg)] containing either galactosylsucrose, xylosylfructoside or sucrose (100 g/kg) or the above fiber-free diet without test sugar (control) for 21 d. Body mass gain was greater in rats fed sucrose, but not in rats fed galactosylsucrose or xylosylfructoside, than in control rats. The mass of the small intestine and colon plus rectum was larger in rats fed xylosylfructoside than in control rats. Cecal contents and cecal tissue mass were heavier, water content of cecal contents was higher, and pH and ammonia concentration of cecal contents were lower in rats fed diets containing xylosylfructoside than in control rats. Galactosylsucrose had similar effects, although not all differences were significant. The concentration of hydrogen ion in cecal contents positively correlated to total cecal concentration of measured organic acids and to amount of cecal contents. Total concentration of measured organic acids in cecal contents positively correlated to cecal tissue mass. The estimated contribution of galactosylsucrose, xylosylfructoside and sucrose for body mass deposition were 0.19, -0.29 and 0.51 (g body mass gained/g sugar), respectively.
A polyclonal antibody specific for the Amadori compound, a product of an early stage of the Maillard reaction, was raised in rabbits by immunization with hexitol-lysine (1-glucitol-lysine or 1-mannitol-lysine) coupled with various carrier proteins. The affinity purified antibody has a high titre and preferentially recognizes the glucose adduct, in the presence of sodium borohydride, as judged on enzyme-linked immunosorbent assay as well as immunoblot analysis. The glycated proteins (Amadori products) in various tissues of normal and streptozotocin-induced diabetic rats were examined by immunoblot analysis. In diabetic conditions, kidney, liver, lens, brain and lung proteins are more susceptible to glycation than other tissue proteins. Heart, spleen, adrenal gland and muscle proteins exhibit similar extents of glycation in both normal and diabetic conditions. This is the first demonstration of a specific antibody against the Amadori compound being raised with a synthetic compound, and of the tissue distribution of glycated proteins in normal and diabetic conditions. The antibody was very useful for in vitro and in vivo experiments on the Maillard reaction.
Cinacalcet hydrochloride (cinacalcet) is a positive allosteric modulator of the calcium-sensing receptor indicated for the treatment of secondary hyperparathyroidism in dialysis patients. In vitro study has demonstrated that cinacalcet is a potent inhibitor of cytochrome P450 (CYP) 2D6 with a K(i) value of 0.087 micromol/L, which is comparable to the well-known potent CYP2D6 inhibitor, quinidine (0.064 micromol/L). A clinical study was conducted to assess the inhibitory effect of cinacalcet on CYP2D6 substrates in healthy volunteers. Each subject received 50 mg of cinacalcet or a matched placebo orally once daily for 8 days with 30 mg of dextromethorphan coadministered on day 8. The mean AUC(0-infinity) and C(max) of dextromethorphan increased 11- and 7-fold, respectively, in extensive metabolizers when coadministered with cinacalcet versus placebo. Therefore, during concomitant treatment with cinacalcet, it may be necessary to consider making dose adjustments for drugs with a narrow therapeutic index that are mainly metabolized by CYP2D6.
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