Mutations of the human thyrotropin-beta subunit glycosylation site reduce thyrotropin synthesis independent of changes in glycosylation status.

In recent studies, site-directed mutagenesis has been used to alter the tripeptide glycosylation recognition sequences of glycoprotein hormone subunits, thereby affecting their structure and function. However, it is not known whether these effects result from changes in glycosylation status, amino acid sequence, or both. We therefore studied the synthesis of wild-type and mutant recombinant human thyrotropins produced by transient transfection of a human cell line. Mutating the TSH-beta subunit glycosylation recognition sequence, Asn-Thr-Thr (codons 23-25), to either Gln-Thr-Thr or Asn-Thr-Tyr abolished subunit glycosylation, as demonstrated by the inability to incorporate 3H-carbohydrates. However, a third mutation (Asn-Thr-Ser) contained an intact glycosylation recognition sequence site, and was shown to retain glycosylation. The mutations that abolished TSH-beta subunit glycosylation resulted in greater than 90% decreases in TSH synthesis. However, the glycosylation recognition sequence mutant that retained beta subunit glycosylation exhibited a 70% decrease in TSH production. These decreases were not attributable to the intracellular accumulation of TSH or its free beta subunit. We also engineered two TSH-beta subunit mutations that did not alter the glycosylation recognition sequence. A glycine to arginine mutation adjacent to the glycosylation recognition sequence, in a region thought to be critical for heterodimer formation, abolished TSH production. In contrast, shortening the TSH-beta subunit carboxyterminus by six amino acids increased TSH synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of naloxone on the preoperative gastric volume and pH

The effect of 4 mg oral naloxone on preoperative gastric volume and pH of gastric aspirate was studied in a double-blind, randomized study. Twenty patients received 10 ml of naloxone (4 mg) mixed with 10 ml of orange juice, and 20 patients received 10 ml of isotonic saline mixed with 10 ml of orange juice, 2 h before surgery. Gastric content was obtained immediately after intubation of the trachea. No significant difference in gastric volume and pH of gastric aspirate was found between the two groups. It is concluded that naloxone does not affect gastric emptying and gastric acid secretion to a degree great enough to protect against aspiration of gastric contents into the lungs.     

Alpha-ketoacids stimulate rat renal cysteine conjugate beta-lyase activity and potentiate the cytotoxicity of S-(1,2-dichlorovinyl)-L-cysteine

Renal cysteine conjugate beta-lyase (beta-lyase) catalyzes the bioactivation of nephrotoxic cysteine S-conjugates. beta-Lyase activity is present in both renal cytosolic and mitochondrial fractions, and, although the cytosolic beta-lyase is identical to glutamine transaminase K, the mitochondrial beta-lyase has not been characterized. Because beta-lyase is a pyridoxal phosphate (PLP)-dependent enzyme, pyridoxamine phosphate (PMP) formation may occur during the metabolism of cysteine S-conjugates. In this study, the effects of alpha-ketoacids, which may convert the PMP form of the enzyme to the pyridoxal phosphate form, on the metabolism and cytotoxicity of cysteine S-conjugates were examined; the PMP enzyme is catalytically inactive in beta-elimination reactions, but is catalytically active in transamination reactions. Both alpha-keto-gamma-methiolbutyrate (KMB) and alpha-ketobutyrate enhanced the metabolism of S-(2-benzothiazolyl)-L-cysteine (BTC) to 2-mercaptobenzothiazole by rat renal cytosol or mitochondria. KMB and phenylpyruvate potentiated both the cytotoxicity of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) in isolated rat renal proximal tubular cells and the inhibition of mitochondrial respiration produced by DCVC. These results are consistent with the formation of PMP during the renal cytosolic or mitochondrial metabolism of cysteine S-conjugates. Mitochondrial beta-lyase was previously localized in the outer membrane. To examine whether beta-lyase activity is present in mitoplasts, but in the PMP form, the effects of KMB on the metabolism of BTC to 2-mercaptobenzothiazole and on the DCVC-induced inhibition of state 3 respiration in mitoplasts were studied. The majority of the mitochondrial beta-lyase activity was present in the outer membrane, and the specific activity of the outer membrane beta-lyase was greater than that of the mitoplast beta-lyase. KMB produced equivalent stimulation of beta-lyase activity in intact mitochondria, in mitochondrial outer membranes, and in mitoplasts and potentiated DCVC-induced inhibition of respiration in intact mitochondria, but not in mitoplasts. These results provide additional evidence for the central role of beta-lyase in the bioactivation of nephrotoxic cysteine S-conjugates.