Silage studies IX biochemical changes in microbe-free silage

Summary If the aseptic plant material is regarded as a substrate for preservative microbial activity, it may be said that both the nitrogen and carbohydrate components undergo changes due to the influence of native enzymes in the plant material. These changes may very well be reflected in the microbial activity in a silage.It is interesting to compare the findings of this investigation, which show that during the days immediately after ensilage practically no changes take place when no bacteria are present. In a non-sterile ensilage, on the other hand, it is during this early stage that the briskest bacterial activity takes place.The chances of, say, an acid-forming microbial process actually lowering the pH in order that preservation of the material may take place are directly counteracted by the action of native enzymes of the vegetable material. During the days immediately after ensilage, however, if other conditions are favourable, microbial activity may clearly determine the whole course of events, and may possibly even inactivate the processes of the plant material itself.As is evident from the experiments described above, the activities inherent in the ensiled material itself are of no mean proportions. Further study of the mutual relationship of the ensilage and the various microbial fractions is therefore particularly necessary in order to be able to understand a number of processes of fundamental importance in biological preservation of plant material.     

Ontogeny of hormone-secreting cells of the rat pituitary gland: An immunocytochemical study on dissociated cells

Summary An immunocytochemical study was undertaken in foetal, prepubertal and mature rats to determine the time of differentiation of various types of adenohypophyseal cells during development. Freshly dissociated pituitary cells from foetal (18–21 days postconception), neonatal (from birth up to 30 days) and adult rats (more than 8 weeks) were characterized using immunocytochemical methods. All types of hormone-producing cells were present at day 18 postconception, although only 20% of the cells were immunolabelled. Adrenocorticotropin (ACTH)-secreting cells accounted for the highest number of hormone-positive cells. Growth hormone-secreting cells increased remarkably from day 18 postconception onwards. Prolactin-secreting cells were not seen in the foetal adenohypophysis and did not start to increase until 10 days after birth, whereas by that time the number of ACTH, thyrotropin, follicle-stimulating and luteinizing hormone-secreting cells had stopped increasing. By day 30 after birth, 80–95% of the cells were immunoreactive.     

Butyrophilin,an apical plasma membrane-associated glycoprotein characteristic of lactating mammary glands of diverse species

Lipid globule membranes were isolated from human and bovine milk and from the milk of sheep, goat, pig, rat and guinea pig, and their polypeptide compositions were analyzed. The major polypeptides with molecular weights similar to that of bovine butyrophilin were separated by gel electrophoresis, isolated and characterized with respect to isoelectric point, molecular weight, immunological cross-reactivity and peptide composition after proteolytic cleavage. We show that in all species examined these proteins are similar to bovine butyrophilin in (i) their relative insolubility in buffers of low and high ionic strength and in non-denaturing detergents, (ii) the occurrence of several isoelectric variants, and (iii) patterns of peptides obtained by protease digestion. It is concluded that closely related proteins are major constituents of the cytoplasmic coat structures associated with milk lipid globule membranes of many species, and we propose the name butyrophilins for this group of proteins. Bovine and human butyrophilins are glycosylated with relatively large amounts of glucosamine, mannose, glucose and galactose but little fucose, sialic acids or galactosamine. Most if not all of the sugar residues are associated with an acetone-soluble peptide fragment of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 12 000–16 000 focusing at about pH 4.0. We suggest that this fragment contains a membrane-spanning peptide sequence and is involved in the attachment of the cytoplasmic coat to the membrane of the milk lipid globule.     

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.

     

Determination of mercury in human serum and packed blood cells by neutron activation analysis

A method is described for the determination of mercury in human blood serum and packed blood cells employing neutron activation analysis. Great attention was devoted to the collection and manipulation of the samples. The accuracy and precision of the method were tested by analyzing biological reference materials and by comparing the concentrations measured in a number of serum samples to those obtained by another, independent technique (cold vapor atomic absorption spectrometry) in the same samples. The article reports the levels measured in blood serum and packed blood cells samples from 15 adult volunteers, as well as the figures determined in a “second-generation” biological reference material (freeze-dried human serum), prepared and conditioned at the University of Ghent.     

Using XAD resins to study the effects of reduced organic micropollutant concentrations in River Rhine and Meuse water on phytoplankton growth

Laboratory incubation experiments were used to study the effect of reduced concentrations of organic micropollutants in water from the rivers Rhine and Meuse on the specific growth rate of the river phytoplankton community. Before incubation, part of the water sampled was treated with XAD-4 and XAD-8 resins to absorb dissolved organic compounds. Four dilutions were made by mixing untreated water with XAD-treated water in the ratios 100:0 (control), 70:30, 40:60 and 0:100. The phytoplankton specific growth rate increased significantly with the increased fraction treated with XAD in all but one incubation experiment. In these experiments, the specific growth rate was on average 9% higher in the fraction in which 100% was treated with XAD than in the controls. In the Rhine and Meuse river water, phytoplankton growth seemed to be inhibited by organic compounds. This inhibition was ascribed to the presence of dissolved organic micropollutants. Removing organic micropollutants using XAD resins to study the toxic effects of these compounds on field phytoplankton communities can be concluded to be a promising tool for risk assessment of micropollutants but needs to be supported by additional methodological research.     

Study of the influence of plasmids on the arbitrary primer polymerase chain reaction fingerprint of Escherichia coli strains

Abstract To study the effect of plasmids on the arbitrary primer-polymerase chain reaction fingerprint of bacterial strains, the Escherichia coli strains DH5, Top10, and W3110 were transformed with plasmids of different sizes: respectively, pUC19, pCEP and two clinically important plasmids carrying resistance to several antibiotics. Total DNA, i.e. both chromosomal and plasmid DNA, was prepared from transformed cells by boiling the cell suspensions and by phenol-chloroform extraction; chromosomal DNA was prepared by the same methods from the non-transformed, plasmid-free strains; plasmid DNA of pUC19 was purchased; plasmid DNA of pCEP was purified from the transformed strains by caesium chloride density gradient centrifugation. Arbitrarily primed polymerase chain reaction was carried out for all of these preparations. Amplification carried out independently with three different primers resulted in similar patterns for the chromosomal preparations whether or not plasmid was present. Amplification of plasmid DNA gave different patterns, characterized by fragments larger than those obtained when total or chromosomal DNA were used as the target. These data illustrate that the plasmids studied here do not influence the chromosomal arbitrarily primed PCR fingerprint, although plasmids alone are amplified in the absence of chromosomal DNA. Experiments comparing different relative concentrations of plasmid and chromosomal DNA indicate that under natural conditions the amount of chromosomal DNA per cell is sufficient to inhibit observable amplification of the plasmid(s) present.     

Dynamics and attenuation characteristics of periphyton upon artificial substratum under various light conditions and some additional observations on periphyton upon Potamogeton pectinatus L.

The seasonal variation in periphyton dynamics has been studied upon artificial substratum (microscopic glass slides) under various light conditions during the periods May–October 1986 and May–September 1987, in Lake Veluwe. Some additional observations on the periphyton development upon leaves of Potamogeton pectinatus L. have been made simultaneously. Four different light conditions were created in an experimental setup by manipulating the photon flux density through artificial shading.Periphyton upon artificial substratum exhibited a relatively high abundance with a distinct seasonal pattern. Periphyton accrual rates were highest at the beginning of June and in August and September upon slides which were incubated for two weeks. Periphyton mass increased during May and June, decreased or remained about the same during July and subsequently increased until an upper plateau was reached upon slides which were incubated from the beginning of May onwards.Generally, periphyton mass was lower upon slides than upon P. pectinatus. The seasonal variation in periphyton mass was more pronounced upon P. pectinatus leaves than upon the slides.Attenuation by periphyton upon slides ranged from 5 to 65% after two weeks of incubation. Periphyton upon slides which had been incubated for more than two weeks demonstrated an attenuation of more than 85%.Water quality parameters other than photon flux density were probably more important in determining the periphyton dynamics, since only minor differences were observed in periphyton mass between the various light conditions. Chlorophyll-a content was higher with increased shading on various sampling dates.Periphyton, especially older periphyton consisted largely of settled silt and clay particles and to a lesser extent of detrital matter on both substrata. Living epiphytes were only a relatively small fraction.It is concluded that a reduction of resuspension of sediment particles, giving less suspended matter in the water column, will result in lower periphytic mass. Consequently, the quantity of photosynthetically active radiation reaching the submerged macrophytes is expected to increase considerably.