Elektronenmikroskopische Untersuchungen an den Hüllen der Oozyten und Eier des Flussbarsches Perca fluviatilis

Zusammenfassung Die Hüllen von Oozyten und reifen Eiern des Fußbarsches wurden licht- und elektronenmikroskopisch untersucht. In Oozyten vor der Bildung der Zona radiata sind die meisten der keulenförmigen Mikrovilli gerade auf die Follikelzellen ausgerichtet. Gelegentlich verlaufen Mikrovilli auch parallel zur Oozytenoberfläche. Die Zona radiata reiferer Eizellen besteht aus zwei Schichten, der elektronendichten Zona radiata externa und der weniger dichten Zona radiata interna. Während die erstere aus einem hochorganisierten Material besteht, ist die letztere aus einer Substanz von geringerer Dichte zusammengesetzt. In der Matrix der Zona radiata interna liegen unregelmäßig angeordnete flaschenbürstenähnliche Einschlüsse. Nach Kontrastierung mit l%iger Phosphorwolframsäure und 0,5% igem Uranylazetat sind feine Fibrillen in der Matrix nachweisbar. Die Zona radiata externa ist außen von einer Gallerthülle umgeben. Sie ist etwa fünfmal so stark wie die gesamte Zona, radiata. Diese Gallerthülle besteht aus homogenem Material von geringerer Dichte. Nach Anfärbung mit 0,2%-igem Rutheniumrot färbt sich diese Schicht intensiv rot. Daraus läßt sich auf das Vorhandensein von sauren Mukopolysacchariden schließen. In der Gallerthülle liegen elektronendichte Körnchen, in denen sich bei hoher Auflösung eine hochorganisierte, kristalloide Struktur nachweisen läßt. Die Gallerthülle wird von den zytoplasmatischen Fortsätzen der kegelförmigen Follikelzellen durchdrungen. Diese Fortsätze gehen in Ausläufer über, die in die Porenkanäle der Zona radiata eindringen. In einigen Fällen umhüllen die Ausläufer der Follikelzellen die Mikrovilli der Oozyte vollständig. Normalerweise liegen die Mikrovilli und die Ausläufer aber deutlich voneinander getrennt. Die Mikrovilli enden entweder direkt an der Zellmembran der Follikelzellen oder sie dringen tief in Membraneinstülpungen ein. Mikrovilli und die Ausläufer der Follikelzellen befinden sich in den Porenkanälen fast reifer Eier. Kurz vor der Ovulation werden sie wahrscheinlich zurückgezogen. Die Porenkanäle reifer Barscheier sind im äußeren Drittel der Zona radiata interna verschlossen.

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Summary The envelope of oocytes and mature eggs of the perch was examined by light and electron microscope. In oocytes before the formation of the zona radiata most of the clubshaped microvilli project straightly towards the follicular cells. Some, however, run parallel to the oocyte surface. The zona radiata of more mature oocytes consists of the electron dense zona radiata externa and the less electron dense zona radiata interna. While the former consists of a highly organized material, the latter is composed of a substance of low density, with the exception of a few brush-shaped inclusions which are scattered irregularely within the matrix of the zona radiata interna. After treatment with 1% phosphotungstic acid and 0.5% uranyl acetate tiny fibres become visible. External to the zona radiata externa the oocytes are enveloped by a jelly layer, about five times as thick as the zona radiata. The jelly layer appears to consist of a rather homogeneous material of low density. After treatment with a solution of 0,2% ruthenium red the jelly layer is stained intensely red, suggesting the presence of acidic mucopolysaccharides. Within the matrix of the jelly layer are embedded electron dense granules of unknown function. At high resolution they reveal a well organized, crystalloid structure. The jelly layer is penetrated by cytoplasmic processes of the cone-shaped follicular cells. These processes develop extensions which enter the pore canals of the zona radiata. In some cases these extensions envelope the microvilli of the oocytes completely. But normally the microvilli and the extensions are clearly separated. The microvilli make either direct contact with the membrane of the follicular processes or penetrate deeply into membrane interdigitations. Microvilli together with follicular extensions are present in the pore canals of almost mature eggs. They seem to be withdrawn just before ovulation. The pore canals of the zona radiata are partly closed in mature eggs.

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Ich verdanke die Anregung zu dieser Arbeit einer Diskussion, die ich mit Herrn Prof. Dr. E. A. Arndt, Rostock, anläßlich seines Vortrages am 21. Februar 1966 in Kiel führte.

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Untersuchungen mit Unterstützung durch die Deutsche Forschungsgemeinschaft. Herrn Prof. Dr. O. Moritz, Institut für Pharmakognosie, danke ich für die Möglichkeit, das Elektronenmikroskop seines Institutes zu benutzen. Frau R. Bardenhewer, geb. Schmidtke, danke ich für die Hilfe bei der Präparation und für die Anfertigung der photographischen Vergrößerungen.     

Semisynthesis of Arg15, Glu15, Met15, and Nle15-aprotinin involving enzymatic peptide bond resynthesis

The semisynthesis of homologues of aprotinin, the bovine pancreatic trypsin inhibitor, is described. The P₁ lysine¹⁵ residue was replaced by two methods. The first procedure, which consisted of two enzymatic steps for the incorporation of other amino acids has previously been described. The second approach consisted of six steps of both enzymatic and chemical nature. The modified inhibitor, in which the lysine¹⁵-alanine¹⁶ peptide bond is hydrolyzed, was used as the starting material. All carboxyl groups of the modified inhibitor were esterified with methanol; the lysine¹⁵ methylester group was then selectively hydrolyzed. Afterward, lysine¹⁵ itself was split off. Arginine, glutamic acid, methionine, andl-2-aminohexanoic acid (norleucine, Nle) were incorporated using water-soluble carbodiimide combined with an acylation catalyst. The methylester group was used to prevent polymerization. The reactive-site peptide bonds were resynthesized using either chymotrypsin or trypsin.     

Cu(I)-thionein release from copper-loaded yeast cells

Summary The release of intact CU(I)₈-thionein from copper-resistant copper-loaded yeast cells, strain X 2180-1Aa, has been shown. This copper(I)-thiolate-rich protein was characterized and compared with the chemical and physicochemical properties of intracellular yeast Cu-thionein. The same molecular mass and stoichiometry of 8 mol copper atoms/mol protein was found. No detectable difference between the Cu-thioneins was seen in luminescence emission, electronic absorption in the ultraviolet region, chiroptical data or amino acid composition. The importance of stable Cu(I)-thiolates in Cu-thionein as a safe vehicle for transporting copper in a non-reactive manner is confirmed.     

Direct Evidence That Excitotoxicity in Cultured Neurons Is Mediated via N-Methyl-D-Aspartate (NMDA) as well as Non-NMDA Receptors

Cultured GABAergic cerebral cortex neurons were exposed to the excitatory amino acid (EAA) L-glutamate, kainate (KA), N-methyl-D-aspartate (NMDA), or RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionate (AMPA). To ensure a constant glutamate concentration in the culture media during the exposure periods, the glutamate uptake inhibitor L-aspartic acid beta-hydroxamate was added at 500 microM to the cultures that were exposed to glutamate. Each of these EAAs was able to induce neurotoxicity. It was not possible to reduce or prevent glutamate-induced cytotoxicity by blocking only one of the glutamate receptor subtypes with either the NMDA receptor antagonist D-(-)-2-amino-5-phosphonopentanoate (APV) or with one of the specific non-NMDA antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). However, if the cultures were exposed simultaneously to glutamate and the antagonists in combination, i.e., APV plus CNQX or APV plus DNQX, the toxicity was completely prevented. Furthermore, CNQX and DNQX were shown to be selective blockers of cytotoxic phenomena induced by non-NMDA glutamate agonists with no effect on NMDA-induced cell death. Likewise, APV prevented NMDA-induced cell death without affecting the KA- or AMPA-induced neurotoxicity. It is concluded that EAA-dependent neurotoxicity is induced by NMDA as well as non-NMDA receptors.     

Homogeneous penetration but heterogeneous binding of antibodies to carcinoembryonic antigen in human colon carcinoma HT-29 spheroids

Summary The monoclonal antibodies 38S1, directed against the carcinoembryonic antigen (CEA), were tested for penetration and binding in human colon carcinoma HT-29 spheroids. Penetration was studied with a method which has not previously been used in immunological investigations. The method, which allows unbound substances to be visualized, is based on freeze drying, vapour fixation, dry sectioning and dry autoradiography. The antibodies penetrated easily and all parts of the HT-29 spheroids seemed to be reached within 15 min. The penetration was even faster than in control glioma U-118MG spheroids that did not express CEA. Binding of the 38S1 antibodies was demonstrated after processing with conventional histology and autoradiography. The binding in the HT-29 spheroids was, after a 1-h incubation period, extremely heterogeneous and occurred mainly in the peripheral parts. More cells were binding the antibodies after 8-h and 32-h incubations and these cells were arranged in peripheral clusters. No binding at all was seen in the CEA-negative glioma spheroids. The distribution of CEA antigens in monolayers and in frozen sections of spheroids of HT-29 cells was analysed with immunohistochemical staining using polyclonal CEA antibodies. The CEA antigens were heterogeneously distributed in both spheroids and monolayers and were as heterogeneous as the binding of the monoclonal antibodies in the living spheroids. Thus, the heterogeneous binding in the living spheroids was not due to penetration barriers, but instead to the heterogeneity in the CEA antigen expression.     

Coupled reductive and oxidative degradation of 4-chloro-2-nitrophenol by a co-immobilized mixed culture system

Summary The restriction of oxygen transfer in Ca-alginate beads used for the immobilization of microbial cells was applied to a coupled reductive and oxidative microbial degradation of the xenobiotic 4-chloro-2-nitrophenol (CNP). The conversion of CNP by Enterobacter cloacae under anaerobic conditions led to the formation of 4-chloro-2-aminophenol (CAP, 81%) and 4-chloro-2-acetaminophenol (CAAP, 16%) after 50 h incubation. CAP, the main reduction product, was further degraded under aerobic conditions by Alcaligenes sp. TK-2, a hybrid strain isolated by conjugative in-vivo gene transfer. Whereas both degradation steps excluded one another in homogeneous systems with free cells, a coupled reductive and oxidative degradation of CNP was observed in one aerated reactor system after co-immobilization of both strains in Ca alginate. The diameter of the alginate beads used for immobilization was recognized as one main factor determining the properties of this mixed culture system. Offprint requests to: H.-J. Rehm     

A T7 promoter-specific, inducible protein expression system forBacillus subtilis

The adaptation and application of theEscherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression inBacillus subtilis is reported. The expression cassette used inBacillus subtilis was tightly regulated and T7 RNA polymerase (T7 RNAP) appeared 30 min after induction. The efficiency of T7 promoter-specific gene expression inB. subtilis was studied using one secretory and two cytosolic proteins of heterologous origin. The accumulation ofE. coli -galactosidase, as well as a 1,4--glucosidase fromThermoanaerobacter brockii inB. subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity. The-amylase ofThermoactinomyces vulgaris, a secretory protein, was found to accumulate in the culture supernatant up to levels of about 70 mg/l 10–20 h after T7 RNAP induction, but was also deposited in cellular fractions. The addition of rifampicin inhibited-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation     

Eradication of established hepatic human neuroblastoma metastases in mice with severe combined immunodeficiency by antibody-targeted interleukin-2

 A major problem in the treatment of solid tumors is the eradication of established, disseminated metastases. Here we describe an effective treatment for established experimental hepatic metastases of human neuroblastoma in C. B.-17 scid/scid mice. This was accomplished with an antibody-cytokine fusion protein, combining the unique targeting ability of antibodies with the multifunctional activity of cytokines. An anti-(ganglioside GD2) antibody (ch14.18) fusion protein with interleukin-2 (ch14.18-IL2), constructed by fusion of a synthetic sequence coding for human interleukin-2 (IL-2) to the carboxyl end of the Cγ1 gene of ch14.18, was tested for its therapeutic efficacy against xenografted human neuroblastoma in vivo. The ch14.18-IL2 fusion protein markedly inhibited growth of established hepatic metastases in SCID (severe combined immunodeficiency) mice previously reconstituted with human lymphokine-activated killer cells. Animals treated with ch14.18-IL2 showed an absence of macroscopic liver metastasis. In contrast, treatment with combinations of ch14.18 and recombinant IL2 at dose levels equivalent to the fusion protein only reduced the tumor load. Survival times of SCID mice treated with the fusion protein were more than double that of control animals. These results demonstrate that an immunotherapeutic approach using a cytokine targeted by an antibody to tumor sites is highly effective in eradicating the growth of established tumor metastases. Received: 7 November 1995 / Accepted: 15 December 1995