Synthesis of N-(1-deoxyhexitol-1-yl)amino acids, reference compounds for the nonenzymic glycosylation of proteins

The N-(1-deoxy-D-mannitol-1-yl) and N-(1-deoxy-D-glucitol-1-yl) derivatives of L-valine, L-alanine, L-threonine, and L-leucine were prepared by reductive amination of D-mannose and D-glucose with the appropriate amino acids, in the presence of sodium cyanoborohydride. N epsilon-(1-Deoxy-D-mannitol-1-yl)- and N epsilon-(1-deoxy-D-glucitol-1-yl)-L-lysine were prepared by similar reactions of hexoses with N alpha-tert-butoxycarbonyl and N alpha-benzyloxycarbonyl-L-lysine, followed by removal of the protecting groups. The structures were confirmed by 1H-n.m.r. spectroscopy, which showed that each compound was completely free of its C-2 epimer. The synthetic compounds may be used as reference compounds for the identification of N-(1-deoxyhexitol-1-yl)amino acids formed when N-(1-deoxy-D-fructose-1-yl) groups of nonenzymically glycosylated proteins, of the hemoglobin A1c type, are reduced with sodium borohydride, and the protein is subjected to acid-catalyzed hydrolysis.     

Purification of the serum acid-stable insulin-like growth factor binding protein from the pig (Sus scrofa)

1. An acid-stable IGF binding protein was isolated and purified from porcine serum. 2. The protein comprised two major species with Mrs of 45 and 41 kDa determined using SDS-PAGE under reducing conditions. 3. The IGFBP preparation specifically bound both IGF-I and II. 4. Four distinct protein bands (Mrs of 23, 45, 50 and 75 kDa) in the porcine IGFBP preparation specifically bound radiolabelled IGF-I. 5. The porcine IGFBP exhibited sequence homology with IGFBPs from human plasma and rat serum. 6. This is the first report of the purification and characterization of the acid-stable IGFBP from porcine serum.     

Ligand-induced endocytosis of the EGF receptor is blocked by mutational inactivation and by microinjection of anti-phosphotyrosine antibodies

Early events in ligand-induced endocytosis of the EGF receptor have been examined. A mutant EGF receptor devoid of intrinsic protein-tyrosine kinase activity bound EGF and dimerized normally yet failed to undergo ligand-induced internalization. Immunofluorescence microscopy revealed that receptors lacking kinase activity failed to undergo the ligand-induced internalization characteristic of receptors with kinase activity. Monoclonal anti-phosphotyrosine antibodies effectively inhibited phosphorylation of exogenous substrates in vitro and, when microinjected into cells containing active EGF receptors, prevented internalization of the receptor when cells were subsequently challenged with EGF. These results point to a crucial role for the kinase activity of the EGF receptor in the process of ligand-induced endocytosis of receptors, and imply that a phosphorylated substrate(s) is required.     

Alkaloid production by transformed root cultures of Catharanthus roseus

Transformed roots of Catharanthus roseus were obtained following infection of detached leaves with Agrobacterium rhizogenes. Roots would not grow in full strength Gamborg's B5 medium but would grow satisfactorily if the medium was diluted to one half strength. Little alkaloid appeared in the growth medium but root tissue contained a high level and wide variety of alkaloids. Ajmalicine, serpentine, vindolinine and catharanthine were prominent components. Vinblastine could also be detected by a combination of HPLC and radioimmunoassay, though at a level of only 0.05g/g dry weight.Abbreviations B5 Gamborg's B5 nutrient salts - LC/MS combined liquid chromatography/mass spectrometry - FW fresh weight - Kb kilobase     

Studies on the biosynthesis of tropane alkaloids in Datura stramonium L. transformed root cultures

The relative contributions made by the l-arginine/agmatine/N-carbamoylputrescine/putrescine and the l-ornithine/putrescine pathways to hyoscyamine formation have been investigated in a transformed root culture of Datura stramonium. The activity of either arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17) was suppressed in vivo by using the specific irreversible inhibitors of these activities, dl--difluoromethylarginine or dl--difluoromethylornithine, respectively. It was found that suppression of arginine decarboxylase resulted in a severe decrease in free and conjugated putrescine and in the putrescine-derived intermediates of hyoscyamine biosynthesis. In contrast, the suppression of ornithine decarboxylase activity stimulated an elevation of arginine decarboxylase and minimal loss of metabolites from the amine and alkaloid pools. The stimulation of arginine decarboxylase was not, however, sufficient to maintain the same potential rate of putrescine biosynthesis as in control tissue. It is concluded that (i) in Datura the two routes by which putrescine may be formed do not act in isolation from one another, (ii) arginine decarboxylase is the more important activity for hyoscyamine formation, and (iii) the formation of polyamines is favoured over the biosynthesis of tropane alkaloids. An interaction between putrescine metabolism and other amines is also indicated from a stimulation of tyramine accumulation seen at high levels of dl--difluoromethylornithine.Abbreviations ADC arginine decarboxylase - DFMA dl--dif-luoromethylarginine - DFMO dl--difluoromethylornithine - MPO N-methylputrescine oxidase - ODC ornithine decarboxylase - PMT putrescine N-methyltransferase We are indebted to Dr. E.W.H. Bohme of Merrell Dow Research Laboratories (Cincinnati, Ohio, USA) for kind gifts of DFMO and DFMA and to Dr. M.J.C. Rhodes for helpful advice and discussion.     

Mg2-dependent phosphatidate phosphohydrolase of rat lung: development of an assay employing a defined chemical substrate which reflects the phosphohydrolase activity measured using membrane-bound substrate

An assay of pulmonary phosphatidate phosphohydrolase activity has been developed that employs a chemically defined liposome substrate of equimolar phosphatidate and phosphatidylcholine. Enzyme assays employing this substrate resolved two distinct activities based upon their requirements for Mg2+. Assays were performed in the presence and absence of 2 mM MgCl2 and the Mg2+-dependent phosphatidate phosphohydrolase activity calculated by difference. The Mg2+-independent phosphatase activity resembled that found using aqueous dispersions of phosphatidate (PAaq). Approximately 90% of the Mg2+-dependent phosphatidate phosphohydrolase activity was recovered in the cytosol and the remainder was associated with the microsomal fraction. The Mg2+-dependent phosphatidate phosphohydrolase activity has kinetic parameters of Km = 55 microM, Vmax = 1.6 nmol/min/mg protein for the microsomal fraction, and Km = 215 microM, Vmax = 6.8 nmol/min/mg protein for the cytosolic fraction. These parameters resembled those found using the microsomal membrane-bound (PAmb) substrate. In addition, the pH optima and sensitivity to detergents and thermal inactivation are equal to those for the PAmb-dependent phosphatidate phosphohydrolase activity. In the course of these studies the microsomal and cytosolic activities were qualitatively equal, indicative of a single enzyme in two subcellular locations. In conclusion, the assay of Mg2+-dependent phosphatidate phosphohydrolase activity measured using equimolar phosphatidate and phosphatidylcholine liposomes is equivalent to that activity previously described using microsomal membrane-bound substrate. However, the chemically-defined system provides a more simplified starting point for further studies on this important enzyme.     

Effect of feed and water deprivation or force-feeding on plasma prolactin concentration in turkey hens

Plasma concentrations of prolactin (Prl), glucose, corticosterone, and D(-)-3-hydroxybutyrate (DBHB) were compared in nonlaying, nonincubating turkey hens subjected to feed and/or water deprivation. Neither Prl nor corticosterone concentrations were significantly (P greater than 0.05) altered by any of the treatments, whereas fasting significantly (P less than 0.05) reduced the concentration of glucose and increased the concentration of DBHB. Plasma levels of Prl in incubating hens were significantly (P less than 0.05) reduced by nest deprivation either in the absence of feed and water or when the hens were force-fed the normal intake for a laying hen. After 48 h of nest deprivation, the hens resumed nesting within 5 min of being returned to the pen although the plasma levels of Prl were low. Neither nest attentiveness nor the concentration of Prl were affected by force-feeding the hens while they were incubating eggs. The concentration of glucose increased in response to force-feeding or nest deprivation, whereas the concentration of corticosterone was increased only by force-feeding. These results suggest that Prl may not be involved in the striking changes in both intermediary and water metabolism which occur during incubation in the turkey hen. Furthermore, since incubation behavior can occur in the presence of low concentrations of Prl, elevated levels of Prl during broodiness appear to be maintained by a stimulus associated with the nest itself or some other aspect(s) of the environment.