Refined 2 A X-ray crystal structure of porcine pancreatic kallikrein A, a specific trypsin-like serine proteinase. Crystallization, structure determination, crystallographic refinement, structure and its comparison with bovine trypsin

Porcine pancreas kallikrein A has been crystallized in the presence of the small inhibitor benzamidine, yielding tetragonal crystals of space group P4₁2₁2 containing two molecules per asymmetric unit. X-ray data up to 2·05 Å resolution have been collected using normal rotation anode as well as synchrotron radiation. The crystal structure of benzamidine-kallikrein has been determined using multiple isomorphous replacement techniques, and has subsequently been refined to a crystallographic R-value of 0·220 by applying a diagonal matrix least-squares energy constraint refinement procedure.Both crystallographically independent kallikrein molecules 1 and 2 are related by a non-integral screw axis and form open, heterologous “dimer” structures. The root-mean-square deviation of both molecules is 0·37 Å for all main-chain atoms. This value is above the estimated mean positional error of about 0·2 Å and reflects some significant conformational differences, especially at surface loops. The binding site of molecule 1 in the asymmetric unit is in contact with residues of molecule 2, whereas the binding site of the latter is free and accessible to the solvent. In both molecules the characteristic “kallikrein loop”, where the peptide chain of kallikrein A is cleaved, is only partially traceable. The carbohydrate attached to Asn95 in this loop, although detectable chemically, is not defined.A comparison of the refined structures of porcine kallikrein and bovine trypsin indicates spatial homology for these enzymes. The root-mean-square difference is 0·68 Å if we compare only main-chain atoms of internal segments. Remarkably large deviations are found in some external loops most of which surround the binding site and form a more compact rampart around it in kallikrein than in trypsin. This feature might explain the strongly reduced activity and accessibility of kallikrein towards large protein substrates and inhibitors (e.g. as shown by the model-building experiments on inhibitor complexes reported by Chen &; Bode. 1983).The conformation of the active site residues is very similar in both enzymes. Tyr99 of kallikrein, which is a leucyl residue in trypsin, protrudes into the binding site and interferes with the binding of peptide substrates (Chen &; Bode. 1983). The kallikrein specificity pocket is significantly enlarged compared with trypsin due to a longer peptide segment, 217 to 220, and to the unique outwards orientation of the carbonyl group of cis-Pro219. Further, the side-chain of Ser226 in porcine kallikrein, which is a glycyl residue in trypsin, partially covers Asp 189 at the bottom of the pocket. These features considerably affect the binding geometry and strength of binding of benzamidine.     

Na+, Cl− cotransport in Ehrlich ascites tumor cells activated during volume regulation (regulatory volume increase)

Ehrlich ascites cells were preincubated in hypotonic medium with subsequent restoration of tonicity. After the initial osmotic shrinkage the cells recovered their volume within 5 min with an associated KCl uptake. The volume recovery was inhibited when NO-3 was substituted for Cl-, and when Na+ was replaced by K+, or by choline (at 5 mM external K+). The volume recovery was strongly inhibited by furosemide and bumetanide, but essentially unaffected by DIDS. The net uptake of Cl- was much larger than the value predicted from the conductive Cl- permeability. The undirectional 36Cl flux, which was insensitive to bumetanide under steady-state conditions, was substantially increased during regulatory volume increase, and showed a large bumetanide-sensitive component. During volume recovery the Cl- flux ratio (influx/efflux) for the bumetanide-sensitive component was estimated at 1.85, compatible with a coupled uptake of Na+ and Cl-, or with an uptake via a K+,Na+,2Cl- cotransport system. The latter possibility is unlikely, however, because a net uptake of KCl was found even at low external K+, and because no K+ uptake was found in ouabain-poisoned cells. In the presence of ouabain a bumetanide-sensitive uptake during volume recovery of Na+ and Cl- in nearly equimolar amounts was demonstrated. It is proposed that the primary process during the regulatory volume increase is an activation of an otherwise quiescent, bumetanide-sensitive Na+,Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump, stimulated by the Na+ influx through the Na+,Cl- cotransport system.     

High-resolution color video cytophotometry

Comparison was made between cytophotometric measurements obtained using two data acquisition systems, one a microphotometer and the other a rapid video camera system, to ascertain whether the degradation of data with the faster video acquisition system still results in recorded images of sufficient quality to permit computer discrimination between cells of very similar appearance. Normal-appearing intermediate cells from cases with normal cytology and those from patients with dysplasia or malignant disease, as well as the subvisual markers within these cells that have rendered them capable of cytophotometric discrimination, were used for the study. Comparison of the data recorded by the two systems indicates that the diagnostic information is preserved in the change-over to a full-field, video-rate scanning system, with differences in the data caused primarily by differences in the spectral response of the two systems. This was reflected in the substantial differences observed in the color-related features and the lesser differences seen in the textural features, while the morphometric features (outline and shape) were virtually unaffected. The differences were primarily expressed on a cell-to-cell basis; in sets of about 300 cells, which would be used in patient-to-patient comparisons, the feature values showed remarkable consistency between the two systems.     

Comparison of the localization of nucleic acid synthesis during bud formation on leaf fragments and on intact undetached leaves ofBegonia rex Putz.

The budding capacity ofBegonia rex leaf fragments is well known; that of undetached leaves has been shown by us only recently after treating the leaves with 6γγ DMAAP. Benzyladenine is as effective as 6γγ DMAAP in stimulating budding. Lower temperatures (17°, 22–12°, 12°) are also capable of inducing bud formation but only after a small cut has been made in a main vein of the undetached leaf. Root formation can also be provoked on undetached leaves which have a cut in the main leaf vein by higher temperatures (24–22°) or by an IAA treatment. Differences in the first stages of bud formation on leaf fragments and on undetached leaves are observed using histochemical and histoautoradiographic techniques.     

Mycoplasma-like bodies in plants infected with potato witches' broom disease and the response of plants to tetracycline treatment

The virus origin of a Czechoslovak isolate of potato witches' broom disease is discounted: electron micrographs of ultrathin section of phloem tissues from plants infected with potato witches' broom disease demonstratedMycoplasma-like bodies, spherical or elongated showing binary fission and fragmentation. The minute corpuscles have a diameter of about 50–60 nm, the largest bodies of 1000 nm. The width of elongated filamentous structures is about 200 nm, most oval bodies have a diameter of 250 nm. A weak tetracycline treatment of diseased plants causes a delay of symptom development; a strong dose of tetracycline (applied by means of the wick method into the stem) inhibits symptom appearance completely. Tetracycline produces a phytotoxic effect inhibiting the growth of tomato plants and causing (at higher concentrations) necrosis and death of these plants. There is a note in the paper dealing with the term “mycoplasma”. The word mycoplasma in the sense ofEriksson (1897) or ofMereschkowsky (1910) does not correspond to the genus nameMycoplasma Nowak (1929).     

Etude ultrastructurale de l'endoplasme et des vacuoles chez deux types de Dinoflagellés appartenant aux genres Noctiluca (Suriray) et Blastodinium (Chatton)

Résumé L'ultrastructure des éléments cytoplasmiques de deux Dinoflagellés a révélé des différences fondamentales directement liées à leur mode de vie.Chez Noctiluca (Dinoflagellé libre, incolore), les corps de Golgi jouent un rôle important dans la formation des sphères archoplasmiques au moment des divisions et lors de l'élaboration de l'endoglée. Les mitochondries de grande taille, parfois géantes, ont des crêtes nombreuses et une matrice réduite. Elles offrent souvent des relations de contiguïté avec les granulations lipidiques et l'enveloppe nucléaire; elles sont très abondantes dans le tentacule du trophozoïte. Il n'existe pas de plastes. Les inclusions lipidiques abondent ainsi que les trichocystes fibreux et les poches mucifères dont le contenu est polysaccharidique.Chez Blastodinium (Dinoflagellé parasite, coloré sauf une espèce), les corps de Golgi sont très réduits dans le trophocyte, assez rares mais de structure classique dans les sporocytes. Les mitochondries sont de petite taille, pauvres en crêtes et ont une matrice importante chez le trophocyte; elles sont plus grandes dans les sporocytes. Les plastes bien développés, périphériques, possèdent des lamelles stromatiques formées de trois thylakoïdes chez les sporocytes; par contre, chez l'individu végétatif, le plus souvent incolore et relativement protégé de la lumière par plusieurs rangées de sporocytes, les plastes, de grande taille ont un aspect dégénéré: lamelles stromatiques rares, thylakoïdes tassés et stroma important; ce sont des étioplastes. Le cas de Blastodinium contortum hyalinum toujours incolore, est particulier: les plastes existent encore mais sont totalement dégénérés. Il y a très peu de lipides dans le trophocyte, mais ils sont par contre abondants dans les sporocytes. Il existe des trichocystes fibreux et chez l'individu végétatif, des figures myéliniques.Ces importantes variations structurales illustrent parfaitement, dans l'échelle évolutive des Dinoflagellés, l'exemple de deux modes de vie très différents.

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Fine structure of endoplasm and vacuoles in two types of dinoflagellates of the genus Noctiluca and Blastodinium

Summary The fine structure of cytoplasmic elements of two Dinoflagellates reveals fundamental differences directly linked to their way of life.In Noctiluca, free-living, colourless dinoflagellate, the Golgi bodies play an important role in the formation of archoplasmic spheres at the moment of division and when the endoglea is elaborated. The mitochondria of great size, sometimes giant, have very abundant cristae and a reduced matrix. They often show relations of contiguity with the lipid granules and the nuclear envelope; they are very abundant in the tentacle of the trophozoïd. There are no plastids. The lipid inclusions are very abundant and so are fibrous trichocysts and muciferous bags whose content is polysaccharidic.In Blastodinium, parasitic dinoflagellate, coloured except for one species, the Golgi bodies are much reduced in the trophocyte, rather rare but of classical structure in the sporocytes. The mitochondria are of small size, with rare cristae and have an enlarged matrix in the trophocyte; they are bigger in the sporocytes. The plastids are well developed, peripheral, and have stromatic lamellae formed of three thylakoïds in the sporocytes. On the other hand, in the vegetative individual, usually colourless since it is protected from the light by many rows of sporocytes, the large plastids have a degenerated aspect, rare stromatic lamellae, packed thylakoïds and enlarged stroma; they are etioplastids. The case of Blastodinium contortum hyalinum, always colourless, is special: the plastids still exist but are completely degenerated. There are very few lipids in the trophocyte but abundant ones in the sporocytes. Some fibrous trichocysts occur, and in the vegetative individual, some myelinic vacuoles.These important structural variations illustrate very well the example of two very different ways of life, in the evolutionary tree of dinoflagellates.

Cet article fait partie d'un travail d'ensemble constituant une Thèse de Doctorat d'Etat intitulée: La cytologie et la division de deux types de Dinoflagellés: Noctiluca Suriray (genre libre) et Blastodinium Chatton (genre parasite).     

Phosphorus disturbances associated with potato leafroll virus infection

Potato leafroll virus induces considerable disturbances of phosphorus metabolism in leafroll-infected potato plants. Each variety reacts differently in this respect on the infection, to some extent being dependent on the susceptibility of the respective variety to leafroll and to the season as well. In varieties particularly susceptible to leafroll as in Apta and Sieglinde there is a decrease of total P after infection. On the contrary, varieties showing medium susceptibility such as varieties Tatranka, Rita and Ambra react to leafroll infection with a considerable increase of total P. In the comparatively less susceptible variety Krasava, there is no change in the level of total P after infection. In autumn, however, a slight decrease of total P even in the medium susceptible variety Tatranka, and the less susceptible variety, Krasava, can be found. Our results shed a new light on existing controversies in literature regarding phosphorus content in leafroll-infected potato plants. When the changes in the contents of various phosphorus fractions in the infected plants are compared we can observe a tendency of lowmolecular fractions to rise. Among the different phosphorus fractions one common feature could be seen, except for the hypersensitive resistant variety Apta, namely, that the acid-soluble organic phosphates in the stems of all varieties examined, were considerably higher than in healthy stems.     

Enzymbestimmungen in Erythrocyten bei Kindern mit Down-Syndrom

Zusammenfassung Bei 20 Kleinkindern mit gesicherter freier Trisomie 21 wurden die Aktivitäten von 21 Enzymen des Energie- und des Glutathionstoffwechsels in isolierten Erythrocyten gemessen. Die Aktivitäten der Fructose-6-phosphatkinase, der Lactatdehydrgenase, der Glucose-6-phosphatdehydrogenase, der Glutathionreduktase (NADP) und der Glutamat-Oxalacetat-Transaminase wurden gegenüber gleichaltrigen Kontrollen signifikant erhöht gefunden. Lediglich die etwa 50%ige Steigerung der Fructose-6-phosphatkinase-Aktivität kann im Sinne eines Gen-Dosis-Effektes gedeutet werden.

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Enzyme activities in the erythrocytes of children with Down's syndrom

Summary The activities of 21 enzymes of the energy supplying and the glutathione metabolism were estimated in isolated erythrocytes from children with Down's syndrome aged from 1 to 5 1/2 years. All 20 patients were trisomic for chromosome 21. The following enzymes were estimated by means of optic assays: All glycolytic enzymes, two enzymes of the hexosephosphate shunt and two of the citric cycle, NADP- and NAD-dependent glutathione reductase, glutamic-oxaloacetic transaminase, adenylate kinase, and Mg²⁺-activated ATPase.The activities of five enzymes, i.e. phosphofructokinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, glutathione reductase (NADP), and glutamic-oxaloacetic transaminase, were significantly elevated as compared to age-matched controls. The 50% increase of phosphofructokinase-activity is assumed to provide suggestive evidence for a controlling gene-locus on chromosome 21. The remaining findings are not considered to be directly related to the chromosomal abnormality.

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Direcktor: Prof. Dr. H.-R. Wiedemann

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Nach einem Vortrag auf der 10. Tagung der Gesellschaft für Anthropologie und Human-genetik, Königstein (Ts.), 22.–25. 10. 1967.

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Die Untersuchungen wurden durch die dankenswerte Unterstützung der Deutschen Forschungsgemeinschaft ermöglicht.