Role of phospholipase A2 in the stimulation of sponge cell proliferation by homologous lectin

Using the Geodia cydonium system, we showed that after incubation of competent sponge cells in the presence of lectin, phospholipase A2 was released from the cells. The substrates for this enzyme, phosphatidylethanolamine and phosphatidylcholine, were identified in the extracellular material of sponge tissue. In addition, the phospholipase A2 inhibitor calelectrin was identified by immunobiochemical techniques; this molecule was associated with the aggregation factor. Reconstitution experiments strongly suggested that phospholipase A2 catalyzed the release of arachidonic acid, which is then taken up by the cells. Intracellularly, arachidonic acid was metabolized primarily to prostaglandin E2. Inhibition studies revealed that prostaglandin E2 is involved in the ultimate increase of DNA synthesis. These findings suggest that the phospholipase A2-arachidonic acid system is involved in the matrix-initiated signal transduction pathway in sponges.     

Resolution of d,l-menthol by interesterification with triacetin using the free and immobilized lipase of Candida cylindracea

Summary Interesterification in isooctane with triacetin as an acyl donor was found to be a new and effective method of racemic resolution of d,l-menthol, when using the free and immobilized lipase of Candida cylindracea. No water was produced by this highly stereoselective type of reaction in contrast to ester synthesis with acetic acid as an acyl donor. Even with diacetin no possible back reaction occurred and the enzyme was easily separated from the reaction solution as opposed to ester hydrolysis in aqueous systems. Inhibition of interesterification was caused by increasing concentrations of the acyl donor triacetin by more than 10 mmol·l^-1 on the one hand, and especially by diacetin on the other hand. The reaction product menthyl acetate had no influence. By adding water the interesterification activity of the lipase was reduced significantly. An alteration of the acyl donor triacetin to longerchained triglycerides caused changes in higher specific activities but poor enantioselectivities of the products, as in the case of ester synthesis starting from longer-chained organic acids.Dedicated to Prof. Dr. Fritz Wagner on the occasion of his 60th birthday     

A non-alphoid repetitive DNA sequence from human chromosome 21

Summary A non-alphoid repetitive DNA from human chromosome 22, consisting of a 48-bp motif, shows homology to both G-group chromosomes in the gorilla, thus indicating the presence of additional repeat family members on further human chromosomes. Therefore, we screened a chromosome-21-specific cosmid library using this repetitive sequence from chromosome 22 (D22Z3). Some 40–50 cosmid clones were positive in tests for hybridization. One of the clones giving the strongest signals was digested with EcoRI/PstI, which we knew to cut frequently within the repeats; this resulted in fragments containing repeat units only. The fragments were subcloned into plasmid vector pTZ 19. Sequence-analysis of a 500-bp insert showed ten copies of a 48-bp repeat similar to D22Z3, with about 15% sequence deviation from the chromosome 22 consensus sequence. In situ hybridization of the newly isolated recombinant established its chromosome 21 specifity at high stringency. Physical mapping by pulsed field gel electrophoresis placed this new repeat in close vicinity to the chromosome 21 alphoid repeat. No cross-hybridization with other mammalian genomes except for those of apes was observed. The locus has been designated D21Z2 by the Genome Data Base. A gel mobility shift assay indicated that this repetitive motif has protein-binding properties.     

Degradation of aniline and monochloroanilines by Rhodococcus sp. An 117 and a pseudomonad: a comparative study

Two newly isolated aniline-degrading bacterial strains were characterized with regard to their enzyme systems responsible for aniline catabolism. One of them identified as a Rhodococcus sp. metabolized aniline exclusively via the beta-ketoadipate pathway by means of inducible enzymes. The aniline-degrading enzyme system of the second isolate, presumably a pseudomonad, was shown to consist of an inducible aniline-converting enzyme and constitutive meta-pathway enzymes. Both isolates failed to metabolize monochlorinated anilines in the absence of additional carbon sources. To explain this the ring-cleaving enzymes of both isolates were examined for their substrate specificities. Furthermore, the effect of 4-chlorocatechol on the enzymes catalyzing aniline conversion and catechol oxygenation was investigated.     

Versuche zur Frage der Erhaltungszüchtung beim Kulturchampignon

Zusammenfassung 1. Die Arbeit befaßt sich mit Fragen der Erhaltungszüchtung beim Kulturchampignon. Von den drei Möglichkeiten der Vermehrung, Mycelteilung, Gewebekultur und Aussaat, wird die Mycelteilung untersucht.2. Es wurden zu diesem Zweck Vielsporkulturen (viele Kerntypen) und Einsporkulturen (nur 2 Kerntypen) miteinander verglichen.3. Eine Ein- und eine Vielsporkultur wurden bei Kultur auf Agar-Nährboden fortlaufend durch Mycelteilung vermehrt. Während die Einsporkultur auch nach 40maliger Teilung keine Mycelveränderung zeigte, traten bei der Vielsporkultur nach 13maliger Vermehrung Degenerationserscheinungen in Form von langsam wachsendem belagartig anliegendem Mycel auf. Später bildete sich teilweise wieder schneller wachsendes Mycel.In Ertragsprüfungen zeigte die Vielsporkultur in der höchsten geprüften Vermehrungsstufe (27. V.) einen starken Ertragsabfall. Bei der Einsporkultur trat kein Ertragsrückgang ein.4. Beide Stämme wurden auch durch Überimpfungen von Körnern vermehrt. Nach achtmaliger Vermehrung war kein Ertragsabfall nachzuweisen. Der in einem zweiten Versuch mit der Einsporkultur allein festgestellte Ertragsrückgang nach 15 Vermehrungen konnte nicht statistisch gesichert werden.5. Von einer Schale der 22. Vermehrung der Vielsporkultur, die belagartige langsamwachsende und fädige schnellwachsende Sektoren enthielt, wurden von beiden Mycelarten Stücke abgeimpft. Es war dadurch möglich, die verschiedenen Wuchstypen voneinander zu trennen. Jedoch bildeten sich manchmal wieder fädige Sektoren im belagartigen Typ und umgekehrt. Insgesamt wurde sechsmal hintereinander Mycel der verschiedenen Typen abgeimpft (6 Teststufen).6. Die Trennung der verschiedenen Wuchstypen wird mit Kernentmischung erklärt. Die Frage bleibt offen, ob Kerne vom Typ des langsam wachsenden Mycels schon von der Aussaat an in der Vielsporkultur waren oder erst später durch Mutation oder Modifikation entstanden. In diesem Falle hätten sich die Degenerationserscheinungen genausogut in der Einsporkultur wie in der Vielsporkultur zeigen können.7. Daß die Entmischung unvollkommen war, liegt vermutlich an den großen Mycelstücken, die abgeimpft wurden. In Fortsetzung der Versuche wird mit zerkleinertem Mycel gearbeitet. Dann werden einzelne Zellen, die unter dem Phasenkontrastmikroskop als kernarm bestimmt wurden, übergeimpft.8. Vier Viel- und vier Einsporkulturen wurden in Wachstumstesten bei Auslese auf schnell- und lang-samwachsendes Mycel miteinander verglichen. Es wurden von jedem Stamm aus zehn Kulturschalen die am wenigsten durchsponnene Schale (l-Gruppe) und die am weitesten durchsponnene Schale (s-Gruppe) ausgelesen und vermehrt. Von der l-Gruppe wurde fünfmal hintereinander aus der am wenigsten durchsponnenen Schale, von der s-Gruppe genausooft aus der am weitesten durchsponnenen Schale Mycel auf zehn neue Schalen abgeimpft.Die Differenz zwischen dem durchschnittlichen Myceldurchmesser der l- und s-Gruppe war unterschiedlich und vergrößerte sich nicht mit der Zahl der Überimpfungen. Die l-Gruppen der Vielsporkulturen wuchsen immer langsamer als die s-Gruppen, während es bei den Einsporkulturen auch umgekehrte Fälle gab. Auch war die Differenz zwischen der l-und s-Gruppe im Mycelwachstum bei den Vielsporkulturen doppelt so häufig statistisch gesichert als bei den Einsporkulturen.Für die Erhaltungszüchtung kann daraus gefolgert werden, daß sich Vielsporkulturen bei Vermehrung durch Teilung leichter verändern können als Einsporkulturen.9. Mycelkulturen auf drei verschiedenen Nährböden (Biomalz-Agar, Weizen-Agar und Kompost-Agar) zeigten je nach Nährboden unterschiedliches Wachstum. Auf Biomalz-Agar wuchsen die Kulturen am langsamsten. Auf Kompost-Agar, dem nährstoff-reichsten der drei Nährböden, waren die Unterschiede im Aussehen des Mycels am deutlichsten ausgeprägt.Es wird über einige Arbeiten anderer Autoren berichtet, in denen ein großer Einfluß des Nährbodens auf das Mycel festgestellt wurde.Die Ursachen dieses Einflusses werden diskutiert.Für die Erhaltungszüchtung wird gefolgert, daß die Stammkulturen auf einem möglichst nährstoffreichen Substrat, z. B. Kompost, gehalten werden sollten.10. In Untersuchungen des schlecht wachsenden belagartigen Mycels auf Krankheitsbefall konnten weder Bakterien noch Schadpilze nachgewiesen werden. Die Virusteste (Fusionsversuche, Wärmeschock) fielen unterschiedlich aus. Das Material wird noch mit Hilfe der Elektronenmikroskopie genauer untersucht werden. Über das Ergebnis wird später berichtet.

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Experiments on maintenance of strains of the cultivated mushroomI. Propagation by mycelium transfer

Summary 1. The paper deals with problems of maintaining strains of the cultivated mushroom. There are three possibilities for propagation: Transfer of mycelium, tissue culture and multispore culture. Of these the transfer of mycelium was investigated.2. Multispore cultures (many types of nuclei) and monospore cultures (only two types of nuclei) were compared.3. Mono- and multispore cultures were continuously propagated on agar media by transfers of mycelium. After 40 transfers the monospore culture showed no change in the mycelium. In the multispore culture degenerative symptoms in the form of slowly growing, matted mycelium appeared after 13 transfers, though the faster growing mycelium reappeared later in some cases. After the last (27th) transfer tested, the multispore culture showed a strong decrease in yield; none was found in the monospore culture.4. Both strains were also propagated by grain transfer. After eight transfers there was no decrease in yield, and the one noticed after 15 transfers in a repeat experiment with the monospore culture proved to be statistically not significant.5. From a petri dish containing, after the 22nd transfer of the multispore culture, matted, slow growing and stringy, fast growing mycelia, pieces of both kinds were taken. Thus it was possible to separate the two types of growth. However, sometimes stringy sectors reappeared in the matted type, and vice versa. Mycelia of the different types were transferred six consecutive times.6. The separation of the various types is explained by a separation of types of nuclei. The question whether nuclei of the type of the slow growing mycelium were already present in the multispore culture before starting the culture or originated later by mutation or modification remains open.In case of a modification or mutation the degeneration symptoms could have occurred in the monospore culture as well as in the multispore culture.7. The reason for an incomplete separation could be the transfer of large pieces of mycelium. In further experiments disintegrated mycelium will be used. Then single cells selected under phase contrast microscope as poor in nuclei will be transferred.8. Four multi- and four monospore cultures were compared for growth rates by selection for fast and for slow growing mycelium. For each strain the least overgrown (l-group) and the most overgrown (s-group) were chosen from ten culture plates and subcultured by five successive transfers to ten fresh plates.The difference in average diameter of mycelium from the l-group and s-group varied and did not increase with the number of transfers. The mycelium of the l-groups of multispore cultures always grew slower than that of the s-groups; in monospore cultures the opposite also occurred. The difference between the l- and s-group in the multispore culture was twice as often significant as in the monospore culture. From this one can conclude that, in propagation with frequent transfer, multispore cultures change more easily than do monospore cultures.9. Mycelium cultures on three different media (biomalt agar, wheat agar, compost agar) showed different growth rates. The slowest growth was found on biomalt agar. On compost agar, richest in nutrients, the difference in appearance of the mycelium was most obvious. Papers by other authors who found a great influence of the medium on the mycelium and the reasons for this influence are discussed. For the maintenance of strains original cultures should be kept on a substrate rich in nutrients, i.e. compost.10. Tests for bacteria and fungi on slow growing, matted mycelium were negative. Tests for virus (fusion tests, heat shock) showed different results. Results of electron microscopic studies on this material will be reported later.

     

Technetium and rhenium complexes of mercapto-containing peptides 1. Tc(V) and Re(V) complexes with mercaptoacetyl diglycine (MAG2) and X-ray structure of AsPh4[TcO(MAG2)]·C2H5OH

The reaction of mercaptoacetyl diglycine (MAG₂) with technetium(V) gluconate in aqueous solution produced [TcO(MAG₂)]⁻. A single X-ray structure determination was carried out for the tetraphenylarsonium salt. The dark brown crystals are monoclinic, space group P2(1)/n, with a=12.478(5), b=14.922(5), c=17.183(9) Å and Z=4. The [TcO(MAG₂)]⁻ ion has a square pyramidal geometry with the technetium atom displaced by 0.756 Å towards the oxo ligand from the plane formed by the equatorial S,N,N,O atoms. The rhenium complex AsPh₄[ReO(MAG₂)] was prepared analogously starting from Re(V) gluconate and characterized.     

Transformation of difluorinated phenols by Penicillium frequentans Bi 7/2

The Penicillium frequentans strain Bi 7/2, using phenol as a sole source of carbon and energy,transformed the fluorinated phenols 2,3-, 2,4-, 2,5-and 3,4-difluorophenol rapidly. After growth on phenol, resting mycelia of the fungus converted the difluorophenols completely at an initial concentration of 0.5 mM within 6 hours. The corresponding difluorinated catechols were found to be intermediates of all difluorophenols investigated. A relatively unspecific phenol hydroxylase catalyzed this hydroxylation step and showed activities towards all difluorophenols tested. One difluorocatechol was formed from each difluorophenol substituted with fluorine in the ortho-position, whereas two catechols were formed from 3,4-difluorophenol, due to its two vacant ortho-positions. A partial defluorination (50-77%) was observed in all cases. This revised version was published online in August 2006 with corrections to the Cover Date.     

Immunohistochemical localization of bioactive peptides and amines associated with the chromaffin tissue of five species of fish

Biogenic peptides and amines associated with the chromaffin tissue in Atlantic cod (Gadus morhua), rainbow trout (Oncorhynchus mykiss), European eel (Anguilla anguilla), spiny dogfish (Squalus acanthias) and Atlantic hagfish (Myxine glutinosa) were identified utilizing immunohistochemical techniques. Within the posterior cardinal vein (PCV) in cod, trout and eel, a subpopulation of chromaffin cells displayed immunoreactivity to tyrosine hydroxylase (TH) and dopamine--hydroxylase (DH) but not to phenylethanolamine-N-methyltransferase (PNMT). TH-like immunorectivity was observed within cells in hagfish hearts. Nerve fibres displaying vasoactive intestinal peptide (VIP) immunoreactivity and pituitary adenylyl cyclase activating peptide (PACAP) immunoreactivity innervated cod, trout and ell chromaffin cells. In eel, neuropeptide Y (NPY)-like and peptide YY (PYY)-like immunoreactivity was located within cells in the PCV, including chromaffin cells. Serotonin-like immunoreactivity was observed within eel and cod chromaffin cells and in hagfish hearts. In the dogfish axillary bodies, nerves displaying TH-like, VIP-like, PACAP-like, substance P-like and galanin-like immunoreactivity were observed. These results are compared with those of other vertebrates, and potential roles for these substances in the control of catecholamine release are suggested.