Thyroglobulin interactions with thyroid plasma membranes. The existence of specific receptors and their potential role.

Thyroglobulin binds to isolated thyroid plasma membrane preparations. Binding is pH- and temperature-dependent with 10-fold better binding at pH 5.0 and 37 degrees C than at 0 degrees C and pH 6.0 through pH 7.5. Binding is, however, maximal in 90 min at all pH values and temperatures examined. Although salts can inhibit or enhance thyroglobulin binding depending on the temperature or pH, conditions approaching those of the physiological state are not inhibitory; physiological conditions do inhibit thyrotropin binding to the same membrane preparations. 125I-Labeled thyroglobulin binding is poorly reversed by unlabeled thyroglobulin at all pH values and temperatures studied; excess unlabeled thyroglobulin can, however, readily prevent binding. At pH values greater than 6.0 and at 0 degrees C, the iodine content of thyroglobulin can affect binding, and the 27 S thyroid iodoprotein is relatively ineffective in preventing the binding of the 19 S species. At pH 5.0 and 37 degrees C, there is no difference in binding of highly and less iodinated thyroglobulin, and the 27 S thyroglobulin iodoprotein is effective in preventing 19 S thyroglobulin binding. The complex nature of these results is interpreted in the light of additional data which show (i) that the thyroid membrane recognizes asialothyroglobulin and (ii) that at pH 5.0 and 37 degrees C a membrane-associated neuraminidase is activated which removes sialic acid from thyroglobulin. Vibrio cholerae neuraminidase can substitute for the endogenous neuraminidase. The receptor on thyroid membranes for asialothyroglobulin is similar to the asialoglycoprotein receptor on liver membranes (Morell, A.G., Gregoriadis, G., Scheinberg, I.H., Hickman, J., and Ashwell, G. (1971) J. Biol. Chem. 246, 1461-1467) in that sialic acid on the receptor is critical for receptor expression. It is distinct from the liver asialoglycoprotein receptor in its binding specificity and in its sensitivity to different bacterial and mammalian neuraminidase preparations. Relationships between thyroglobulin and thyrotropin receptors on thyroid membranes are explored, and the functional role of the thyroglobulin receptor is discussed.     

A new approach (cyano-transfer) for cyanogen bromide activation of Sepharose at neutral pH,which yields activated resins,free of interfering nitrogen derivatives

A new approach for the reaction of Sepharose with cyanogen bromide is described, using triethylamine as a “cyano-transfer” reagent. An optimized procedure for activation at neutral pH was developed. This procedure requires only about 5% of the usual amount of cyanogen bromide. Activated resins are free of imidocarbonates and carbamates, containing only active cyanate esters. Extremely high coupling capacities (75 μmol ligand/g wet Sepharose 4B) can be obtained using this method.     

Changes in serum amylase and its isoenzymes after whole body irradiation.

A study was carried out to assess the effect of total body irradiation on pancreatic and parotid isoenzymes of amylase in patients about to undergo bone-marrow transplantation who had received high-dose cyclophosphamide. Twelve patients were studied, enzyme activity being measured before and at various times after total body irradiation. Serum total amylase activity rose rapidly within 12 hours of irradiation to a maximum at 36 hours, returning to normal by six days; most of the increase was derived from salivary damage, with a much smaller pancreatic component. These results confirm that radiation produces acute changes in amylase activity, which may be of use in assessing radiation-induced damage.     

Thyroid membrane ADP ribosyltransferase activity. Stimulation by thyrotropin and activity in functioning and nonfunctioning rat thyroid cells in culture

Bovine thyroid membranes possess both ADP ribosyltransferase and NAD glycohydrolase activities with the same Km values for NAD and the same pH optima. In intact membranes, the ADP ribosyltransferase is limited in its extent by the amount of available membrane acceptor which can be ADP-ribosylated; in membranes solubilized with lithium diiodosalicylate, an artificial acceptor, L-arginine methyl ester, can be substituted to eliminate this limitation. The product of the ADP ribosyltransferase is a mono-ADP-ribosylated acceptor whether the intact or solubilized membrane provides the enzyme activity and whether membrane or exogenous acceptor, L-arginine methyl ester, is utilized. The intact membranes and the solubilized preparation also have an enzyme activity which can release AMP from the mono-ADP-ribosylated acceptor whether formed by the action of the membrane ADP ribosyltransferase or the A promoter of cholera toxin. The NAD glycohydrolase activity appears to represent the half-reaction of the ADP ribosyltransferase, i.e. an activity measurable substituting water for a membrane acceptor or L-arginine methyl ester. Membranes from functional rat thyroid cells in culture, i.e. cells chronically stimulated by thyrotropin and unresponsive to further additions of thyrotropin, have low ADP-ribosylation but high NAD glycohydrolase activities. In contrast, membranes from nonfunctional rat thyroid cells, i.e. cells unresponsive to thyrotropin, have high ADP-ribosylation and low NAD glycohydrolase activities. NAD hydrolysis by the NAD glycohydrolase activity cannot account for the low ADP-ribosylation activity in membranes from the functioning cells, and its low level of ADP-ribosylation can be eliminated by solubilizing the membranes and substituting an artificial acceptor, L-arginine methyl ester. The ADP ribosyltransferase activity of rat thyroid cell membrane preparations can be enhanced by thyrotropin in a dose-dependent manner but not by insulin, glucagon, hydrocortisone, adrenocorticotropin, or its glycoprotein hormone analog, human chorionic gonadotropin. It is thus suggested (i) that, in analogy to cholera toxin, thyrotropin-stimulated ADP-ribosylation may be important in the regulation of the adenylate cyclase response and (ii) that the level of membrane acceptor available for ADP-ribosylation may relate both to a stable "'activated" state of the adenylate cyclase system in cells chronically stimulated with thyrotropin and/or to a desensitized state with regard to a failure of more thyrotropin to elicit additional functional responses.     

Protein-associated DNA breaks in cells treated with adriamycin or ellipticine

The effect of intercalating agents on mammalian DNA in vivo was examined by the technique of alkaline elution. Adriamycin and ellipticine were found to produce large numbers of single-strand breaks. These breaks appeared to be intimately associated with protein to the extent that enzymatic deproteinization of the DNA was necessary to reveal the breaks. The frequency of breaks and cross-links increased with concentration and time of exposure to the drugs. These data suggest that DNA single-strand scission may be a feature common to intercalators. The association with a cellular protein is previously undescribed and suggests possible mechanisms for the strand scission.     

On the chemical basis of chromosome banding patterns.

A comparative study of the staining characteristics of four reagents for human chromosomes has been carried out. The four reagents are: (I) quinacrine mustard, as an alkylating agent, (II) the dihydroxy derivative of quinacrine mustard, (III) quinacrine, and (IV) 9-amino-6-chloro-2-methoxyacridine. The last reagent does not possess the amino substituted side chain even though it has the same intercalating nucleus. Comparison of the first three compounds in their staining and banding behavior suggested the initial step leading to banding may be the displacement of the nucleoprotein sites in hcromosomes. The Q and G banding could be blocked experimentally by treating the chromosome preparation with dimethylamine solution. This result may suggest that these sites have weaker basic proteins (nonhistone proteins?). The use of compound IV, which does not have the side chain in the molecule but does have the same intercalating chromophore, did not lead to banding and gives indirect support to this hypothesis. A combined use of compound IV and quinacrine may be useful for the determination of total DNA vs. banding DNA.     

Divergence in fitness and evolution of drug resistance in experimental populations of Candida albicans

The dissemination and persistence of drug-resistant organisms in nature depends on the relative fitness of sensitive and resistant genotypes. While resistant genotypes are expected to be at an advantage compared to less resistant genotypes in the presence of drug, resistance may incur a cost; resistant genotypes may be at a disadvantage in the absence of drug. We measured the fitness of replicate experimental populations of the pathogenic yeast Candida albicans founded from a single progenitor cell in a previous study (L. E. Cowen, D. Sanglard, D. Calabrese, C. Sirjusingh, J. B. Anderson, and L. M. Kohn, J. Bacteriol. 182:1515-1522, 2000) and evolved in the presence, and in the absence, of the antifungal agent fluconazole. Fitness was measured both in the presence and in the absence of fluconazole by placing each evolved population in direct competition with the drug-sensitive ancestor and measuring the reproductive output of each competitor in the mixture. Populations evolved in the presence of drug diverged in fitness. Any significant cost of resistance, indicated by reduced fitness in the absence of drug, was eliminated with further evolution. Populations evolved in the absence of drug showed more uniform increases in fitness under both conditions. Fitness in the competition assays was not predicted by measurements of the MICs, doubling times, or stationary-phase cell densities of the competitors in isolation, suggesting the importance of interactions between mixed genotypes in competitions.     

An evaluation of the vitamin D3 content in fish: Is the vitamin D content adequate to satisfy the dietary requirement for vitamin D?

It has been suggested that the major source of vitamin D should come from dietary sources and not sun exposure. However, the major fortified dietary source of vitamin D is milk which often does not contain at least 80% of what is stated on the label. Fish has been touted as an excellent source of vitamin D especially oily fish including salmon and mackerel. Little is known about the effect of various cooking conditions on the vitamin D content in fish. We initiated a study and evaluated the vitamin D content in several species of fish and also evaluated the effect of baking and frying on the vitamin D content. Surprisingly, farmed salmon had approximately 25% of the vitamin D content as wild salmon had. The vitamin D content in fish varied widely even within species. These data suggest that the tables that list the vitamin D content are out-of-date and need to be re-evaluated.