Influence of chain shortening on the inhibitor properties of hirudin and eglin c

To find out minimal sizes of the proteinase inhibitor proteins hirudin and eglin necessary for their biological activity the inhibitors were incubated with exopeptidases. From the incubation mixtures shortened derivatives were isolated and characterized. Eglin c can be N-terminally shortened by up to 6 amino-acid residues without any loss of affinity towards chymotrypsin. The complex of thrombin with hirudin lacking 3 C-terminal amino-acid residues showed a 15-20-fold increased Ki value as found previously for desulfato-hirudin and desulfato-hirudin shortened by 2 amino-acid residues. Obviously, the C-terminal part of the hirudin molecule has a positive influence on its affinity to thrombin.     

Interaction of site specific hirudin variants with alpha-thrombin

The kinetics of complex formation between recombinant hirudin or recombinant hirudin mutants with thrombin were analyzed. In order to elucidate the inhibitor's reactive site peptide bond predetermined amino acid substitutions were introduced at positions of basic amino acid residues by means of site-directed mutagenesis of a hirudin gene. In comparison to recombinant hirudin (Ki = 19 pM) only those mutant inhibitors which were modified at amino acid position Lys47 showed a higher Ki value for their complexes with thrombin. The observed effects are mainly due to increased koff rate constants.     

Expression, secretion and processing of hirudin in E. coli using the alkaline phosphatase signal sequence

A DNA fragment coding for the E. coli phoA signal peptide was synthesized and inserted into the expression vector pKK223-3. A single HindIII restriction site is located just at the end of the signal sequence. A gene coding for the proteinase inhibitor hirudin, which has previously been synthesized, was inserted into this HindIII site. The hybrid protein was expressed under control of the tac-promoter and secreted into the periplasm of E. coli. From the periplasmic fraction two processed proteins were isolated. One of these was identical with desulfatohirudin and also had similar biological properties.     

The isolation of lysosomes from normal rat liver by affinity chromatography

Lysosomes from normal rat liver were isolated by affinity chromatography using Sepharose-bound Ricinus communis agglutinins I + II. Characterization of the lysosomal fraction by marker enzymes showed--compared with the homogenate--an enrichment in: acid phosphatase and arylsulfatase about 30- to 60-fold, the tartrate-sensitive acid phosphatase about 95-fold, whereas beta-D-glucosidase, beta-D-galactosidase and sphingomyelinase showed a much higher enrichment of 170- to 260-fold. Marker enzymes for other cell organelles were not detectable. The phospholipid pattern and optical control with electron microscopy gave further indications that the isolated fractions were very rich in lysosomes. A comparison of the phospholipid compositions of plasma membranes isolated from normal rat liver and membranes from the isolated fractions of lysosomes, showed that they were quite different; in particular bis(monoacylglycero)phosphate, which we found to be a typical lysosomal phospholipid, was absent in plasma membranes.     

Dynamics of the cytoskeleton in Amoeba proteus

Fluorescein-labeled muscle actin was microinjected into Amoeba proteus and followed during intracellular redistribution by means of the image-intensification technique. The fully polymerization-competent protein becomes part of the endogenous actomyosin system undergoing dynamic changes over time periods of several hours. Single-frame analysis of long-term sequences enabled the direct demonstration of both the contractile activities and morphological transformations of microfilaments in normally locomoting, immobilized and phagocytozing specimens. In normally locomoting cells the filament layer undergoes continuous changes in spatial distribution depending on the actual pattern of cytoplasmic streaming and cell shape. The highest degree of differentiation is always maintained in the intermediate region between the front and the uroid, thus indicating this segment of the cortex to be the most important site in generating motive force for pseudopodium formation and ameboid movement. In immobilized cells contracted by the application of ruthenium red or relaxed by different anesthetics, the filament layer forms a continuous thick sheath beneath the cell surface or becomes completely disintegrated. In phagocytozing cells the local polymerization of actin at the tip of pseudopodia forming the food-cup and around the nascent phagosome points to a significant participation of the actomyosin system in the process of capturing and constricting prey organisms. Although our results provide clear evidence for the overall importance of motive force generation according to the hydraulic pressure theory, some motile phenomena exist in Amoeba proteus that cannot exclusively be explained by this mechanism.     

Echoortung bei der Fledermaus Chilonycteris rubiginosa

Zusammenfassung Chilonycteris rubiginosa erzeugt in allen Orientierungsituationen dreiteilige Ortungslaute. Im Anfangsteil steigt die Frequenz um etwa 1–2 kHz an. Der folgende Mittelteil hat eine konstante Frequenz von etwa 57 bis 57,6 kHz. Im Endteil fällt die Frequenz um etwa 8 kHz ab. Die Laute werden in Folgen von Lautgruppen ausgesendet.CR erzeugt pro Flügelschlag eine Lautgruppe. Im freien Flug zeigt CR Gruppen mit 2 Lauten von etwa 17–23 msec Dauer. Landende Fledermäuse senden in der Annäherungsphase Gruppen mit einer zunehmenden Zahl immer kürzerer Laute und in der Schlußphase eine längere Gruppe mit vielen kurzen Lauten.Fliegende Tiere senken die Frequenz des konstantfrequenten Mittelteils immer um etwa den Betrag der durch die Fluggeschwindigkeit bedingten Dopplereffekte ab, so daß die von den Tieren gehörte Echofrequenz nahezu konstant in Höhe der vor dem Flug ausgesendeten Frequenz gehalten wird.CR zeigt Kopf- und Ohrbewegungen. Die Ohrbewegungen stehen in Beziehung zur Lautaussendung.

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Echolocation by the bat Chilonycteris rubiginosa

Summary Chilonycteris rubiginosa (CR) produces tripartite ultrasonic sounds in all orientation situations. During the first part the frequency rises by 1–2 kHz. The following middle part has a constant frequency of about 57–57,6 kHz. In the terminal part the frequency decreases by about 8 kHz. The sounds are emitted as a sequence of groups of sounds.In flight they produce per wingbeat one group of sounds at a repetition rate of 10–11 Hz. In free flight CR emits groups of 2 sounds of about 17–23 msec duration. During the approach landing bats emit groups consisting of an increasing number of sounds of decreasing duration. During the terminal phase the group is longer in duration and consists of many short sounds.Flying CR lower the frequency of the middle part by an amount which compensates for Doppler shifts caused by the flight velocity. The frequency heard by the bats is, thus, always kept constant and equal to a frequency which is about 100–150 Hz above the medium frequency emitted before the flight. CR shows head and ear movements. The ear movements are correlated to the sound emission.

     

Regeneration from intact and sectioned immature embryos of barley (Hordeum vulgare L.): the scutellum exhibits an apico-basal gradient of embryogenic capacity

Immature zygotic embryos from spring barley cv. Dissa were used to induce somatic embryogenenesis. Up to 158 germinated somatic embryos could be recovered per plated zygotic embryo. Critical factors for obtaining a high yield of regenerants were the size of the explant, the level of 2,4-D used for callus induction and the careful division of callus at each subculture. Use of microsections of immature embryos as explants revealed a pronounced gradient of callus formation and embryogenic response across the scutellum. Sections from the scutellar tissue at the coleoptilar end of the embryo gave the most callus and were highly embryogenic. The regeneration response of sectioned explants was comparable to that recovered from intact embryos of similar size.