Strukturanalytische Untersuchungen zur Altersabhängigkeit der Sensibilität

Zusammenfassung Eine Analyse der Meßwerte von Ronge (1943) über die Reizausnutzung durch das Tastsinnes-Nervensystem der Haut zeigt im Zusammenhang mit einer vorausgegangenen Studie (Scharf und Blumenthal, 1967), daß der Reizerfolg in einer transzendenten Fläche höherer Ordnung in Abhängigkeit vom Lebensalter (oder von der Anzahl der Meissnerschen Tastkörperchen pro Hautflächeneinheit) und vom Reizdruck dargestellt werden kann. In Druckrichtung steigt diese Fläche mit zunehmendem Reizdruck nichtlinear an, in Zeitrichtung oscilliert die Fläche dagegen träge um Normwerte, die beim 20jährigen Menschen realisiert sind. Dabei werden die Altersveränderungen der histologischen Hautstruktur offenbar zur Kompensation der altersabhängigen Verminderungen der Zahl der Tastkörperchen ausgenutzt.

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Summary An analysis of the observations on the Reizausnutzung by nerves of touch (Ronge, 1943) connected to a previous study (Scharf and Blumenthal, 1967) shows that the irritation result may be figured by a transcendental plane of higher order as a function of age (or number of Meissner's corpuscles per area skin) and irritation pressure. Along the pressure axis this non-linear plane is increasing non-linear in dependence on ascending pressure, but along the time axis the plane oscillates lazily round about the norm values which are realized in human beings of about 20 years of age. It seems that the age-dependent changes of histological skin structure are utilized to compensate the age-dependent diminution of touch corpuscle number.

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Mit dankenswerter Unterstützung durch einen Forschungsauftrag des Staatssekretariates für das Hochschulwesen der DDR.

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Numerische Rechnung: Tischrechner Mercedes Cellatron R 44 SM, Leitende Med. techn. Ass. Ruth Pieper (Anatomisches Institut Halle). Programmgesteuerter Digitalrechner ZRA 1, Math, techn. Ass. Friedegund Hüther (Institut für Numerische Mathematik, Halle).

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Graphik: Akad. Bildhauer Hellmut Helwin.     

Elektronenmikroskopische Untersuchungen zur Dynamik neurosekretorischer Zellen von Enchytraeus (Oligochaeta)

Zusammenfassung Von lichtmikroskopischen Befunden der Neurosekretion bei dem Oligochaeten Enchytraeus ausgehend, haben wir an zwei elektronenmikroskopisch genau bezeichneten und festgelegten Zellen bzw. Zelltypen, die bereits lichtmikroskopisch charakterisiert worden waren, Untersuchungen über die submikroskopisch faßbare Zelldynamik durchgeführt. Die beiden Arten neurosekretorischer Zellen (Q-Zelle und P-Zellen) sind elektronen-mikroskopisch schon durch ihre Lage zu erfassen. Sie können nicht nur durch die Zellgröße, sondern auch durch ihre Elementargranula, den Aufbau des endoplasmatischen Retikulums und den Golgi-Apparat eindeutig unterschieden werden. Wie schon in den lichtmikroskopischen Untersuchungen wurde die Sekretionsaktivität mit der Amputation ausgelöst. Sowohl in der Q -als auch in der P-Zelle bewirkt die Amputation eine unmittelbare Sekretentleerung. Die darauf einsetzende Phase der Sekretproduktion ist submikroskopisch durch eine erhöhte Zahl von Golgistrukturen in diesen Zellen, durch das deutlich in Erscheinung tretende granuläre endoplasmatische Retikulum und durch eine fortschreitende Vergrößerung und Verdichtung von Lysosomen in beiden Zelltypen gekennzeichnet. Für die Q-Zelle sind weiterhin die Verstärkung des diesen Zelltyp besonders noch charakterisierenden Bereiches von Membranzisternen und die dortige Ribosomenbildung typisch. Auf Grund der Feststellungen wird die Frage der Beziehung einzelner Strukturen in diesen beiden Zelltypen zur Produktion des Neuro-sekrets diskutiert. Die elektronenmikroskopische Untersuchung führte zur Entdeckung eines weiteren Zelltyps, der im Lichtmikroskop bisher nicht erkannt worden war und der sich durch besonderen Reichtum an Mitochondrien und großen Lipoid (?)-Komplexen auszeichnet (M-Zelle). Über seine Bedeutung ist jedoch noch keine Aussage möglich.

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Summary The cytophysiology of two types of neurosecretory cells (Q and P cell) in the brain of the oligochete Enchytraeus was studied at the ultrastructural level. These cell types can be identified by their location, and particularly by the size difference of their elementary granules. Amputation of the last ten segments caused a release of secretory product followed by a phase of renewed production. This was characterized by changes in the endoplasmic reticulum, the Golgi apparatus, and the lysosomes. The role of these structures in the production of neurosecretory material was discussed. Furthermore, a cell type with extraordinarily numerous mitochondria, hitherto unknown in Enchytraeus, was described. Its function has not yet been determined.

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Mit Unterstützung durch die Sächsische Akademie der Wissenschaften zu Leipzig.

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Herrn Prof. Dr. F. Seidel, Marburg, zum 70. Geburtstag gewidmet.     

Radioactive labeling of proteins in cultured postimplantation mouse embryos II. Dose and time dependency

Summary The conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations and fluorography. The aim was to obtain highly radioactively labeled proteins under conditions as physiological as possible. Mouse embryos of Days 8, 10, and 11 of gestation were cultured in Tyrode’s solution. Incubation time and concentration of [³H (or¹⁴C)]amino acids in the culture medium were varied over a broad range. Embryos were prepared with placenta and yolk sac or without any embryonic envelopes. After culturing, the physiologic-morphologic state of the embryos was registered on the basis of several criteria. The radioactivity taken up by the total protein of each embryo was determined and calculated in disintegrations per minute per milligram protein per embryo. To approach our aim, embryos of different developmental stages had to be cultured under different conditions. A good compromise for Day-8, Day-10, and Day-11 embryos was: embryos prepared with yolk sac (opened) and placenta, 150 μCi radioactive amino acids added per milliliter medium, incubation for 4 to 5 h. For maximum labeling of proteins it is advisable to culture Day-10 embryos without embryonic envelopes under particular conditions. This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to the project K1 237/3-2 (Systematic analysis of cell proteins).     

Integration of the Serratia marcescens haemolysin into human erythrocyte membranes

The haemolytic activity of Serratia marcescens is determined by two proteins, ShlA and ShlB. ShlA integrates into the erythrocyte membrane and causes osmotic lysis through channel formation. The conformation of ShlA and its interaction with erythrocyte membranes were studied by determining the cleavage of ShlA by added trypsin. Our results suggest that the conformation of inactive ShlA (from an ShlB- strain) differs from the active ShlA, and that in a hydrophobic environment (detergent or membrane) active ShlA assumes a conformation distinct from that in buffer. Only active haemolysin adsorbed to erythrocytes. ShlA was firmly integrated into the erythrocyte membrane since it was only released under conditions which also dissolved the integral erythrocyte membrane proteins. Moreover, ShlA integrated into 'ghosts' remained there and was not haemolytic when incubated with erythrocytes. From the trypsin cleavage pattern obtained with haemolysin and C-terminally truncated, but still active, haemolysin derivatives integrated into erythrocytes, and sealed and unsealed erythrocyte 'ghosts', we conclude that ShlA is preferentially cleaved by trypsin at a few sites but only from the inside of the erythrocyte. Haemolysin in the erythrocyte membrane forms a water-filled channel and is resistant to trypsin and other proteases.     

Bacillus thuringiensis potency bioassays against Heliothis armigera, Earias insulana, and Spodoptera littoralis larvae based on standardized diets

Bacillus thuringiensis screening programs based on the official potency bioassay using third-instar larvae and on a neonate bioassay were developed for Heliothis armigera, Earias insulana, and Spondoptera littoralis. In these bioassays, the diets were standardized to be suitable, with minor modifications, for feeding of the three lepidopterans. The bioassay protocol was based on determination of the LC50 of the microbial standard HD-1-S-80 in the insects susceptible to B. thuringiensis var. kurstaki strains. This was followed by preliminary screening of B. thuringiensis strains at the LC50 of the B. thuringiensis standard. The B. thuringiensis strains causing 100% mortality at this LC50 in the larvae were selected for potency determinations. The neonate bioassay was suitable for accurate determinations of potencies also in S. littoralis--a representative of insects weakly susceptible to the HD-1 standard. The role of the official and the neonate bioassays in developing microbial control programs is discussed.     

Nucleotide sequences of the fecBCDE genes and locations of the proteins suggest a periplasmic-binding-protein-dependent transport mechanism for iron(III) dicitrate in Escherichia coli.

The fec region of the Escherichia coli chromosome determines a citrate-dependent iron(III) transport system. The nucleotide sequence of fec revealed five genes, fecABCDE, which are transcribed from fecA to fecE. The fecA gene encodes a previously described outer membrane receptor protein. The fecB gene product is formed as a precursor protein with a signal peptide of 21 amino acids; the mature form, with a molecular weight of 30,815, was previously found in the periplasm. The fecB genes of E. coli B and E. coli K-12 differed in 3 nucleotides, of which 2 gave rise to conservative amino acid exchanges. The fecC and fecD genes were found to encode very hydrophobic polypeptides with molecular weights of 35,367 and 34,148, respectively, both of which are localized in the cytoplasmic membrane. The fecE product was a rather hydrophilic but cytoplasmic membrane-bound protein of Mr 28,189 and contained regions of extensive homology to ATP-binding proteins. The number, structural characteristics, and locations of the FecBCDE proteins were typical for a periplasmic-binding-protein-dependent transport system. It is proposed that after FecA- and TonB-dependent transport of iron(III) dicitrate across the outer membrane, uptake through the cytoplasmic membrane follows the binding-protein-dependent transport mechanism. FecC and FecD exhibited homologies to each other, to the N- and C-terminal halves of FhuB of the iron(III) hydroxamate transport system, and to BtuC of the vitamin B12 transport system. FecB showed some homology to FhuD, suggesting that the latter may function in the same manner as a binding protein in iron(III) hydroxamate transport. The close homology between the proteins of the two iron transport systems and of the vitamin B12 transport system indicates a common evolution for all three systems.     

Inhibition of lipopolysaccharide O-antigen synthesis by colicin M

Colicin M inhibits peptidoglycan biosynthesis at the level of the bactoprenyl carrier lipid. Since the synthesis of O-antigen also requires bactoprenyl carrier lipid, the effect of colicin M on O-antigen biosynthesis was studied using a colicin-sensitive strain of Salmonella typhimurium. Determination of O-antigen intermediates by two different methods showed that bactoprenyl-dependent O-antigen biosynthesis was inhibited by colicin M. Synthesis of both O-antigen and peptidoglycan was almost immediately inhibited following colicin addition. This was followed some 20 min later by cell lysis. The only known common step between O-antigen and peptidoglycan synthesis is formation of bactoprenyl phosphate by dephosphorylation of bactoprenyl pyrophosphate. Determination of bactoprenyl phosphates showed an accumulation of bactoprenyl pyrophosphate in colicin-treated cultures. It was concluded that dephosphorylation of the bactoprenyl lipid carrier was inhibited by colicin M, and this in turn prevented both O-antigen and peptidoglycan synthesis.