PRIMARY STRUCTURE OF LOBSTER-MUSCLE ARGININE KINASE: Amino and Carboxyl-terminal Structure of the Enzyme and Complete Alignment of the Cyanogen-Bromide Peptides

The acetylation state of the blocked NH₂-terminal end of arginine kinase was characterized by the occurrence of 1 mol acetyl group/mol protein; a 5-residue segment corresponding to the amino-terminal portion was isolated from a pronase digest of the enzyme and its amino acid sequence determined as N-acetyl Ala-Asx-Ala-Ala-Thr. Arginine kinase is terminated at the carboxyl end by the sequence Lys-Glu-MetOH; this particular 3-residue sequence is repeated three times in the overall structure of the protein and is present in three CNBr fragments. One of these, a peptide of 14 amino acid residues, was identified in the course of this study and its amino acid sequence determined. Its location at the COHO-terminal end of the enzyme was recognized on the basis of investigations carried out with des-MetOH-Glu-arginine kinase, a specific proteolytic derivative. The alignment of the eight CNBr-fragments which constitute the arginine kinase molecule was established according to the sequential and compositional properties of seven unique tryptic methionyl peptides isolated from the whole protein. The alignment was confirmed by using BNPS-skatole fragments of the enzyme as another protein source.     

Synthesis of a metal binding protein designed on the α/β scaffold of charybdotoxin*

The α/β scaffold of the scorpion toxin charybdotoxin has been used for the engineering of a metal binding site. Nine substitutions, including three histidines as metal ligands, have been introduced into the original toxin sequence. The newly designed sequence, 37 amino acids long, has been assembled by solid-phase synthesis and HBTU (2-(1H-benzotriazol-l-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) coupling of Fmoc-protected amino acids. Formation of the three disulfide bonds occurred efficiently and rapidly in the presence of glutathione, and this post-synthesis modification has facilitated the purification task enormously. The process of synthesis and purification was performed in less than a week with an overall 10.2°, yield. Circular dichroism analysis showed that the newly designed protein is folded in a α/β structure, similarly to the parent toxin. Electronic absorption spectroscopy, circular dichroism and gel filtration experiments have been used to show that Cu²⁺ and Zn²⁺ ions bind with high affinity to the newly engineered protein. These results demonstrate that the α/β fold, common to all scorpion toxins, is a very versatile basic structure, tolerant for substitutions and able to present new sequences in a predetermined conformation. The chemical approach is shown to be effective, rapid and practical for the production of novel designed small proteins. © Munksgaard 1995.     

The efficiency of irrigants in removing root canal debris when used with an ultrasonic preparation technique

Summary. Three types of irrigant, 2.6 per cent sodium hypochlorite, water and Solvidont were compared for their ability to remove debris during root canal preparation with ultrasonically operated K files. Twenty-seven freshly extracted human teeth were divided into three groups, and each group prepared using one of the irrigants. The teeth were sectioned longitudinally, stained and photographed for assessment by a ranking system. Canals prepared with sodium hypochlorite were found to have significantly less debris remaining than those prepared with water or Solvidont. No significant difference was found between preparations with water and Solvidont.     

Is Pleasantness of Biscuits and Cakes Related to their Actual or to their Perceived Sugar and Fat Contents?

Perceptions of sugar, fat and moisture contents, as well as their influences on pleasantness were investigated in commercial foods. One-hundred-and-two-normal-weight men rated the “pleasantness”, “flavour intensity”, “moisture”, “sweetness” and “fatness” of 39 different biscuits and cakes. Sugar content was accurately perceived up to a maximum content of about 33% weight/weight. The perception of fat content was less accurate and depended on both fat and sugar contents. High sugar contents seemed to decrease perception of fatness. Pleasantness was influenced mainly by sugar content and less by fat content. Pleasantness was better predicted by rated contents than by actual contents; it was even better predicted by the overall flavour intensity. Preferences for high fat stimuli did not appear to be based on conscious perception of their fat content. We conclude that the classical results obtained with simple experimental stimuli remain valid, as a first approximation, for commercial biscuits and cakes, despite their complex sensory characteristics.     

Ontogeny of bovine neurohypophysial hormone precursors

Vasopressin, MSEL-neurophysin and a glycopeptide, here referred to as copeptin, are three fragments of a common protein precursor processed during axonal transport from hypothalamus to neurohypophysis. Neurohormones and neurophysins purified from 7–9-month-old bovine foetuses have previously been shown to be identical with those found in the adult. Copeptin has now been isolated from 7–9-month and 3-month-old bovine foetuses and chemically characterized. It can be concluded from the nature of the three precursors that the same vasopressin gene is expressed in the adult and the 7–9-month-old foetus.     

Complete amino acid sequence of goose VLDV-neurophysin Traces of a putative gene conversion between promesotocin and provasotocin genes

Goose VLDV-neurophysin (mesotocin-associated neurophysin) has been purified from posterior pituitary glands through molecular sieving on Sephadex G-75 and high-pressure reverse-phase liquid chromatography on Nucleosil C-18 columns. Despite apparent molecular mass of unreduced VLDV-neurophysin measured by polyacrylamide gel electrophoresis with sodium dodecylsulfate appeared near 17kDa, this value fell to 11 kDa after reduction with mercaptoethanol, suggesting the existence of a homodimer. Complete amino acid sequence (93 residues) of goose VLDV-neurophysin has been determined. N- and C-terminal sequences of the protein have been established by Edman degradation (microsequencing) and use of carboxypeptidase Y, respectively. Peptides derived from oxidized or carboxamidomethylated neurophysin by trypsin or staphylococcal proteinase hydrolyses have been isolated by high-pressure liquid chromatography and microsequenced, allowing determination of the complete sequence. Comparison within the vertebrate VLDV-neurophysin lineage, namely goose VLDV-neurophysin to mammalian VLDV-neurophysins and to deduced toad VLDV-neurophysin, reveals a residue insertion between positions 66 and 67 in the nonmammalian VLDV-neurophysins. When goose MSEL-neurophysin (vasotocin-associated neurophysin) and goose VLDV-neurophysin are compared to their bovine counterparts, identical substitutions are found in positions 17 (Asn in both goose neurophysins instead of Gly in both ox neurophysins), 18 (Arg instead of Lys), 35 (Tyr instead of Phe), and 41 (Thr instead of Ala). Identity of the sequences 10-74 in both ox neurophysins has been explained by partial gene conversion between oxytocin and vasopressin genes, and identical substitutions in both goose neurophysins might reveal a similar gene conversion between mesotocin and vasopressin genes in birds.     

Katamnestische Untersuchungen griseofulvinbehandelter Onychomykosen

Insgesamt 359 Onychomykosen, die in der Hautpoliklinik der Freien Universität Berlin mit Griseofulvin behandelt wurden, konnten nachuntersucht werden. Es handelte sich um 160 Onychomykosen in Fingern und um 199 Onychomykosen an Zehen. 114 Finger– und 141 Zehennagelmykosen wurden konservativ mit Griseofulvin behandelt. Die übrigen 46 Finger– und 58 Zehennagelmykosen erhielten Griseofulvin in Verbindung mit der Extraktion der erkrankten Nägel. Bei konservativer Therapie wurden 62% der Fingernagelmykosen und 11% der Zehennagelmykosen geheilt. Bei kombinierter Behandlung waren es 80% Erfolge bei den Fingernägeln und 55% Erfolge bei den Zehennägeln. Der Behandlungserfolg hängt aber nicht nur von der Lokalisation der Mykose sondern auch vom Alter des Patienten und von der Höhe der Griseofulvingesamtdosis ab. Bei konservativer Griseofulvintherapie nimmt der Heilerfolg durchschnittlich um 3,1 % und bei kombinierter Behandlung durchschnittlich um 6,6 % mit je zehn Jahren Alterszunahme ab. Zwischen der Tablettenzahl und dem Heilerfolg besteht nach Aussage des Korrelationskoefflzienten für die konservative Therapie starke Abhängigkeit, d. h. je größer die Griseofulvingesamtdosis, die der Patient erhalten hat, ist, desto größer ist die Quote der geheilten Onychomykosen. Insgesamt 155 der 359 Onychomykosen (43 %) konnten geheilt werden. Von den 86 konservativ geheilten Onychomykosen rezidivierten je 9 innerhalb des ersten und des zweiten Jahres nach Therapieende. Zusammen 26 Rezidive traten bei 69 Heilungen durch kombinierte Griseofulvinbehandlung auf. 14mal innerhalb des ersten, 8mal innerhalb des zweiten und 4mal innerhalb des dritten Jahres nach Kurende. Bei beiden Behandlungsmethoden überwiegen die Rezidive der Zehennagelmykosen. 10 Rezidiven bei Onychomykosen der Zehen stehen 8 Rezidive bei Onychomykosen der Finger nach konservativer Behandlung und 18 Rezidiven der Zehennagelmykosen stehen 8 Rezidive bei Fingernagelmykosen nach kombinierter Therapie gegenüber. Zusammenfassend läßt sich feststellen, daß unter den 155 erfolgreich behandelten Onychomykosen sich 108 Onychomykosen der Finger und 47 Onychomykosen der Zehen befanden. 92 der 108 geheilten Fingernagelmykosen (85 %) blieben wahrend einer Nachbeobachtungszeit von mindestens 18 und maximal 42 Monaten rezidivfrei. Bei den Zehennagelmykosen betrug der Anteil der rezidivfreien 19 von 47, d. h. nur 40 z% Bei insgesamt 44 Onychomykosen kam es also zu einem Rezidiv.