Studies on the biotin-binding site of streptavidin. Tryptophan residues involved in the active site.

Streptavidin, the non-glycosylated bacterial analogue of the egg-white glycoprotein avidin, was modified with the tryptophan-specific reagent 2-hydroxy-5-nitrobenzyl (Hnb) bromide. As with avidin, complete loss of biotin-binding activity was achieved upon modification of an average of one tryptophan residue per streptavidin subunit. Tryptic peptides obtained from an Hnb-modified streptavidin preparation were fractionated by reversed-phase h.p.l.c., and three major Hnb-containing peptide fractions were isolated. Amino acid and N-terminal sequence analysis revealed that tryptophan residues 92, 108 and 120 are modified and probably comprise part of the biotin-binding site of the streptavidin molecule. Unlike avidin, the modification of lysine residues in streptavidin failed to result in complete loss of biotin-binding activity. The data imply subtle differences in the fine structure of the respective biotin-binding sites of the two proteins.     

Development of cell surface saccharides on embryonic pancreatic cells

Using a battery of seven lectin-ferritin conjugates as probes for cell surface glycoconjugates, we have studied the pattern of plasmalemmal differentiation of cells in the embryonic rat pancreas from day 15 in utero to the early postpartum stage. Our results indicate that differentiation of plasmalemmal glycoconjugates on acinar, endocrine, and centroacinar cells is temporally correlated with development and is unique for each cell type, as indicated by lectin-ferritin binding. Specifically, (a) expression of adult cell surface saccharide phenotype can be detected on presumptive acinar cells as early as 15 d in utero, as indicated by soybean agglutinin binding, and precedes development of intracellular organelles characteristic of mature acinar cells; (b) maturation of the plasmalemma of acinar cells is reached after intracellular cytodifferentiation is completed, as indicated by appearance of Con A and fucoselectin binding sites only at day 19 of development; conversely, maturation of the endocrine cell plasmalemma is accompanied by "loss" (masking) of ricinus communis II agglutinin receptors; and (c) binding sites for fucose lectins and for soybean agglutinin are absent on endocrine and centroacinar cells at all stages examined. We conclude that acinar, centroacinar, and endocrine cells develop from a common progenitor cell(s) whose plasmalemmal carbohydrate composition resembles most closely that of the adult centroacinar cell. Finally, appearance of acinar lumina beginning at approximately 17 d in utero is accompanied by differenetiation of apical and basolateral plasmalemmal domains of epithelial cells, as indicated by enhanced binding of several lectin-ferritin conjugates to the apical plasmalemmal, a pattern that persists from this stage through adult life.     

Age-Specific Mortality During the 1918 Influenza Pandemic: Unravelling the Mystery of High Young Adult Mortality

The worldwide spread of a novel influenza A (H1N1) virus in 2009 showed that influenza remains a significant health threat, even for individuals in the prime of life. This paper focuses on the unusually high young adult mortality observed during the Spanish flu pandemic of 1918. Using historical records from Canada and the U.S., we report a peak of mortality at the exact age of 28 during the pandemic and argue that this increased mortality resulted from an early life exposure to influenza during the previous Russian flu pandemic of 1889–90. We posit that in specific instances, development of immunological memory to an influenza virus strain in early life may lead to a dysregulated immune response to antigenically novel strains encountered in later life, thereby increasing the risk of death. Exposure during critical periods of development could also create holes in the T cell repertoire and impair fetal maturation in general, thereby increasing mortality from infectious diseases later in life. Knowledge of the age-pattern of susceptibility to mortality from influenza could improve crisis management during future influenza pandemics.

“The war is over – and I must go” Egon Schiele, 1890–1918.

     

Chemical genetics suggests a critical role for lysyl oxidase in zebrafish notochord morphogenesis

As a result of a chemical genetic screen for modulators of metalloprotease activity, we report that 2-mercaptopyridine-N-oxide induces a conspicuous undulating notochord defect in zebrafish embryos, a phenocopy of the leviathan mutant. The location of the chemically-induced wavy notochord correlated with the timing of application, thus defining a narrow chemical sensitivity window during segmentation stages. Microscopic observations revealed that notochord undulations appeared during the phase of notochord cell vacuolation and notochord elongation. Notochord cells become swollen as well as disorganized, while electron microscopy revealed disrupted organization of collagen fibrils in the surrounding sheath. We demonstrate by assay in zebrafish extracts that 2-mercaptopyridine-N-oxide inhibits lysyl oxidase. Thus, we provide insight into notochord morphogenesis and reveal novel compounds for lysyl oxidase inhibition. Taken together, these data underline the utility of small molecules for elucidating the dynamic mechanisms of early morphogenesis and provide a potential explanation for the recently established role of copper in zebrafish notochord formation.     

Chicken avidin exhibits pseudo-catalytic properties. Biochemical, structural, and electrostatic consequences

Avidin and its bacterial analogue streptavidin exhibit similarly high affinities toward the vitamin biotin. The extremely high affinity of these two proteins has been utilized as a powerful tool in many biotechnological applications. Although avidin and streptavidin have similar tertiary and quaternary structures, they differ in many of their properties. Here we show that avidin enhances the alkaline hydrolysis of biotinyl p-nitrophenyl ester, whereas streptavidin protects this reaction even under extreme alkaline conditions (pH > 12). Unlike normal enzymatic catalysis, the hydrolysis reaction proceeds as a single cycle with no turnover because of the extremely high affinity of the protein for one of the reaction products (i.e. free biotin). The three-dimensional crystal structures of avidin (2 A) and streptavidin (2.4 A) complexed with the amide analogue, biotinyl p-nitroanilide, as a model for the p-nitrophenyl ester, revealed structural insights into the factors that enhance or protect the hydrolysis reaction. The data demonstrate that several molecular features of avidin are responsible for the enhanced hydrolysis of biotinyl p-nitrophenyl ester. These include the nature of a decisive flexible loop, the presence of an obtrusive arginine 114, and a newly formed critical interaction between lysine 111 and the nitro group of the substrate. The open conformation of the loop serves to expose the substrate to the solvent, and the arginine shifts the p-nitroanilide moiety toward the interacting lysine, which increases the electron withdrawing characteristics and consequent electrophilicity of the carbonyl group of the substrate. Streptavidin lacked such molecular properties, and analogous interactions with the substrate were consequently absent. The information derived from these structures may provide insight into the action of artificial protein catalysts and the evolution of catalytic sites in general.