Iminoglycinuria in a child in Czechoslovakia

Summary The child with iminoglycinuria is in our observation. Hyperprolinuria was seen at 5 months by screening program. The child was repeatedly examined in the hospital and was seen last time at 16 months. IQ was 67, in the urine were excessive amounts of glycine, proline a hydroxyproline. In the blood aminoacids were in normal levels. In the child was noticed the same increase of proline in the blood as in the control child of the same age following loading test with L-proline, indicating normal intestinal absoption. Both parents and father's sister's 2-year-old mentally retarded child exhibit excessive glycinuria. The father, his sister, father's father, and grandfather are partialy, congenitaly deaf.     

Peptides and multiple antigen peptides from Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase: preparation, immunogenicity and immunoprotective capacity in C57BL/6 mice.

Four monoepitopic MAPs (MAP A, B, C and E) and one bis-diepitopic MAP B-E derived fromthe primary sequence of Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase, previously tested in BALB/c mice, were examined for their immunogenicity and protective capacity in C57BL/6 mice. Despite multimerization into MAPs, MAP Aand MAP C were poorly immunogenic. In contrast toBALB/c mice, MAP E was non-immunogenic in C57BL/6 mice. Peptide B in the form of MAP B orbis-diepitopic MAPB-E elicited immune responses in C57BL/6 mice that were associated with a significant decrease in worm burden. The MAPs were prepared by the stepwise solid-phase peptide synthesis using Boc/Bzl chemistry, successfully purified on the RP-HPLC column and characterized by RP-HPLC, HPCE and MALDI-TOF MS techniques. A general strategy for MAPs purification is discussed here and the purification of MAP Band MAP E is documented in detail.     

Translation of poly(U) on ribosomes from Streptomyces aureofaciens

Slowly cooled cells of Streptomyces aureofaciens contained mainly tight-couple ribosomes. Maximum rate of polyphenylalanine synthesis on ribosomes of S. aureofaciens was observed at 40°C, while cultures grew optimally at 28°C. Ribosomes of S. aureofaciens differed from those of E. coli in the amount of poly(U) required for maximum synthetic activity. The polyphenylalanine-synthesizing activity of E. coli ribosomes was about 3-times higher than that of S. aureofaciens ribosomes. The addition of protein S1 of E. coli or the homologous protein from S. aureofaciens had no stimulatory effect on the translation of poly(U). In order to localize alteration(s) of S. aureofaciens ribosomes in the elongation step of polypeptide synthesis we developed an in vitro system derived from purified elongation factors and ribosomal subunits. The enzymatic binding of Phe-tRNA to ribosomes of S. aureofaciens was significantly lower than the binding to ribosomes of E. coli. This alteration was mainly connected with the function of S. aureofaciens 50 S subunits. These subunits were not deficient in their ability to associate with 30 S subunits or with protein SL5 which is homologous to L7/L12 of E. coli.     

Stoicheiometry of dicyclohexylcarbodiimide-ATPase interaction in mitochondria

1. The oligomeric dicyclohexylcarbodiimide (DCCD)-binding protein of mitochondrial ATPase was studied using (a) the relationship between [¹⁴C]DCCD binding and inhibition of ATPase activities and (b) the analysis of the kinetics of inhibition. 2. The [¹⁴C]DCCD binding to bovine heart mitochondria is linearly proportional to the inhibition of ATP hydrolysis up to a 50% decrease of the original activity resulting in 0.6 mol DCCD bound covalently to the specific inhibitory site (Hous?t?k, J., Svoboda, P., Kopecký, J., Kuz?ela, S?. and Drahota, Z. (1981) Biochim. Biophys. Acta 634, 331–339) per mol of the fully inhibited enzyme. 3. Kinetics of the inhibition of both the ATPase activity (heart and liver mitochondria) and ADP-stimulated respiration (liver) reveal that 1 mol DCCD per mol ATPase eliminates both the synthetic and the hydrolytic activities. It is inferred that the activity-binding correlation underestimates the number of DCCD-reactive sites. 4. The second-order rate constant of the DCCD-ATPase interaction (k) is inversely related to the concentration of membranes, indicating that DCCD reaches the inhibitory site by concentrating in the hydrophobic (phospholipid) environment. 5. At a given concentration of liver mitochondria, comparable k values are obtained both for the inhibition of ATP hydrolysis (k=5.35·10²M^?1·min^?1) and ADP-stimulated respiration (k=5.67·10²M^?1·min^?1). 6. It is concluded that both the synthetic and the hydrolytic functions of ATPase are inhibited via a common single DCCD-reactive site. This site is represented by one of the several polypeptide chains forming the oligomer of the DCCD-binding protein. The inhibitor-ATPase interaction does not exhibit cooperativity, indicating that the preferential reactivity towards DCCD is an inherent property of the inhibitory site.     

Experimental hypersplenism in mice.

The authors induced experimental hypersplenism in mice with repeated intraperitoneal injections of methylcellulose ("Tylosa", Messrs. Kabl). Detailed haematological tests of the peripheral blood, a cytomorphological examination of bone marrow and spleen from puncture material and histological tests of the bone marrow, spleen, lymph nodes, thymus, liver, kidneys and lung tissue of hypersplenic mice were carried out. Among the haematological results, apart from findings of marked erythrocyto-, leucocyto- and thrombocytopenia, attention is drawn to the finding, not previously described in the literature, of lowered osmotic resistance and elevated phagocytic activity of the leucocytes of hypersplenic mice.     

Impact of an extruded nucleotide on cleavage activity and dynamic catalytic core conformation of the hepatitis delta virus ribozyme

The self-cleaving hepatitis delta virus (HDV) ribozyme is essential for the replication of HDV, a liver disease causing pathogen in humans. The catalytically critical nucleotide C75 of the ribozyme is buttressed by a trefoil turn pivoting around an extruded G76. In all available crystal structures, the conformation of G76 is restricted by stacking with G76 of a neighboring molecule. To test whether this crystal contact introduces a structural perturbation into the catalytic core, we have analyzed approximately 200 ns of molecular dynamics (MD) simulations. In the absence of crystal packing, the simulated G76 fluctuates between several conformations, including one wherein G76 establishes a perpendicular base quadruplet in the major groove of the adjacent P1 stem. Second-site mutagenesis experiments suggest that the identity of the nucleotide in position 76 (N76) indeed contributes to the catalytic activity of a trans-acting HDV ribozyme through its capacity for hydrogen bonding with P1. By contrast, in the cis-cleaving genomic ribozyme the functional relevance of N76 is less pronounced and not correlated with the P1 sequence. Terbium(III) footprinting and additional MD show that the activity differences between N76 mutants of this ribozyme are related instead to changes in average conformation and modified cross-correlations in the trefoil turn.     

Third activity of Bordetella adenylate cyclase (AC) toxin-hemolysin. Membrane translocation of AC domain polypeptide promotes calcium influx into CD11b+ monocytes independently of the catalytic and hemolytic activities

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the alpha(M)beta2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration ([Ca2+]i) in target cells. The CyaA-mediated [Ca2+]i increase in CD11b+ J774A.1 monocytes was inhibited by extracellular La3+ ions but not by nifedipine, SK&F 96365, flunarizine, 2-aminoethyl diphenylborinate, or thapsigargin, suggesting that influx of Ca2+ into cells was not because of receptor signaling or opening of conventional calcium channels by cAMP. Compared with intact CyaA, a CyaA-AC- toxoid unable to generate cAMP promoted a faster, albeit transient, elevation of [Ca2+]i. This was not because of cell permeabilization by the CyaA hemolysin pores, because a mutant exhibiting a strongly enhanced pore-forming activity (CyaA-E509K/E516K), but unable to deliver the AC domain into cells, was also unable to elicit a [Ca2+]i increase. Further mutations interfering with AC translocation into cells, such as proline substitutions of glutamate residues 509 or 570 or deletion of the AC domain as such, reduced or ablated the [Ca2+]i-elevating capacity of CyaA. Moreover, structural alterations within the AC domain, because of insertion of various oligopeptides, differently modulated the kinetics and extent of Ca2+ influx elicited by the respective AC- toxoids. Hence, the translocating AC polypeptide itself appears to participate in formation of a novel type of membrane path for calcium ions, contributing to action of CyaA in an unexpected manner.