Five groups of eight newborn calves were used to study absorption of colostral immunoglobulin G. One feeding of 2 liters of pooled colostrum was given at one of 6, 12, 24, 36, or 48 h after birth. Concentrations of immunoglobulin G in blood plasma and feces were measured by an immunodiffusion technique. Plasma volume and fecal excretion also were measured. When colostrum was given 6 h after birth, 65.8% of the ingested immunoglobulin G appeared in the plasma. This percentage declined rapidly to reach 46.9%, 11.5%, 6.7%, and 6.0% when colostrum was given at the ages of 12, 24, 36, and 48 h. Total fecal immunoglobulin G increased linearly with age. The quantities not recovered from plasma and feces reached a maximum when colostrum was given at 24 or 36 h after birth. Immunoglobulin G can be "lost" to a great extent via routes other than plasma and feces during this time. Quantities of immunoglobulin G measured in plasma represent apparent rather than true absorption.
The recent cloning of several human and monkey UDP-glucuronosyltransferase (UGT) 2B proteins has allowed the characterization of these steroid metabolic enzymes. However, relatively little is known about the structure-function relationship, and the potential post-translational modifications of these proteins. The mammalian UGT2B proteins contain at least one consensus asparagine-linked glycosylation site NX(S/T). Endoglycosidase H digestion of the human and monkey UGT2B proteins demonstrates that only UGT2B7, UGT2B15, UGT2B17, and UGT2B20 are glycosylated. Although UGT2B15 and UGT2B20 contain three and four potential glycosylation sites, respectively, site-directed mutagenesis revealed that both proteins are glycosylated at the same first site. In both proteins, abolishing glycosylation decreased glucuronidation activity; however, the K(m) values and the substrate specificities were not affected. Despite the similarities between UGT2B15 and UGT2B20, UGT2B20 is largely more labile than UGT2B15. Treating HK293 cells stably expressing UGT2B20 with cycloheximide for 2 h decreased the enzyme activity by more than 50%, whereas the activity of UGT2B15 remained unchanged after 24 h. The UGT2B20 protein is unique in having an isoleucine at position 96 instead of an arginine as found in all the other UGT2B enzymes. Changing the isoleucine in UGT2B20 to an arginine stabilized enzyme activity, while the reciprocal mutation in UGT2B15 R96I produced a more labile enzyme. Secondary structure predictions of UGT2B proteins revealed a putative alpha-helix in this region in all the human and monkey proteins. This alpha-helix is shortest in UGT2B20; however, the helix is lengthened in UGT2B20 I96R. Thus, it is apparent that the length of the putative alpha-helix between residues 84 and 100 is a determining factor in the stability of UGT2B enzyme activity. This study reveals the extent and importance of protein glycosylation on UGT2B enzyme activity and that the effect of residue 96 on UGT2B enzyme stability is correlated to the length of a putative alpha-helix.
Mineralocorticoid and glucocorticoid hormones are metabolized as glucuronide conjugates. Using labeled [(14)C]uridine diphosphate glucuronic acid and microsomal preparations from human embryonic kidney 293 cells stably expressing the different human and monkey uridine diphosphate glucuronosyltransferase (UGT)2B enzymes, it is demonstrated that the two human allelic variants UGT2B7H((268)) and UGT2B7Y((268)) conjugate aldosterone, its A-ring reduced metabolites (5alpha-dihydroaldosterone and 3alpha,5beta-tetrahydroaldosterone), and both 5alpha- and 5beta-tetrahydrocortisone epimers. The two variants of UGT2B4 also glucuronidate tetrahydroaldosterone, whereas all enzymes tested were inefficient to produce cortisol glucuronide derivatives. Kinetic analyses reveal that UGT2B7 polymorphisms glucuronidate mineralocorticoids with a 5.5- to 20-fold higher affinity than glucocorticoids. For the first time, a significant difference between the two allelic variants of UGT2B7 is described, because UGT2B7H((268)) possesses an 11-fold higher aldosterone glucuronidation efficiency (ratio Vmax((app.))/Km((app.))) than UGT2B7Y((268)). RT-PCR experiments demonstrate the expression of UGT2B7 in human kidney and in renal proximal tubule epithelial cells, suggesting that mineralocorticoids and glucocorticoids are metabolized in their target tissue. Measurement of aldosterone glucuronidation and normalization with the UGT2B protein contents in monkey tissues demonstrate that liver and kidney glucuronidate this hormone with a similar velocity. Immunohistochemical studies performed in monkey kidney cortex reveal a restrictive expression of UGT2B proteins in the epithelial cells of the proximal tubules. Because expression of the mineralocorticoid receptor was detected in the distal tubule epithelial cells, the present data suggest a two-cell mechanism of aldosterone action and metabolism in the kidney.
The effects of chronic toxic stress on the hypothalamo-pituitary-interrenal (HPI) axis were investigated in yellow perch (Perca flavescens) captured at a reference site (Lake Memphremagog) and two sites contaminated with heavy metals and organic contaminants (Ile Perrot and Iles de la Paix, Lake St. Louis) in spring, summer, and fall. Cortisol secretion of the fish was stimulated in situ by an acute capture stress or by an i.p. injection of 4 IU/100 g body mass of porcine corticotropin (ACTH1-39). The response to both these challenges was lower in perch from the highly contaminated site than in perch from the reference site in the spring but not in summer. In fall, fish from the highly contaminated site had, as in spring, a lower response to ACTH than fish from the reference site. The reduced ability of perch to respond to capture stress or to ACTH indicates that the interrenal tissue in fish from contaminated sites is functionally impaired. Cortisol-impaired fish also had abnormal carbohydrate metabolism. The reduced ability of wild fish from contaminated sites to respond to a standardized ACTH challenge may be used as an early indicator of contamination-induced chronic stress.
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