Determination of ovulation and pregnancy in the marmoset (Callithrix jacchus) by monitoring of urinary hydroxypregnanolone excretion

In Callithrix jacchus the urinary excretion of hydroxypregnanolone (5 alpha-pregnane-3 alpha, 7 alpha-diol-20-one) shows a luteal rise during the ovulatory cycle. This progesterone metabolite can therefore be used as an indication of ovulation, implantation, and subsequently for the persistence of the pregnancy. The excretion can be monitored by high performance thin-layer chromatography (HPTLC) and postchromatographic derivatization after enzymatic hydrolysis of the conjugate. Profiles of urinary hydroxypregnanolone levels are presented and correlated with luteinizing hormone excretion. The method is noninvasive and therefore suited for long-term studies in these monkeys.     

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.     

Extended investigation of the substrate specificity of dipeptidyl peptidase IV from pig kidney.

The substrate specificity of dipeptidyl peptidase IV (dipeptidyl peptide hydrolase, EC 3.4.14.5) from pig kidney was investigated, using a series of substrates, in which the amino-acid residue in position P1, a structural derivative of proline, was altered with respect to ring size and substituents. It was demonstrated that dipeptidyl peptidase IV hydrolyses substrates of the type Ala-X-pNA, where X is proline (Pro), (R)-thiazolidine-4-carboxylic acid (Thz), (S)-pipecolic acid (Pip), (S)-oxazolidine-4-carboxylic acid (Oxa), or (S)-azetidine-2-carboxylic acid (Aze). The ring size and ring structure of the residue in the P1 position influence the rate of enzyme-catalysed hydrolysis of the substrate. The highest kcat value (814 s-1) was found for Ala-Aze-pNA. In contrast, the kcat value for Ala-Pro-pNA is nearly 55 s-1. With all substrates of this series, the rate-limiting step of the hydrolysis by dipeptidyl peptidase IV is the deacylation reaction. Compounds of substrate-like structure, in which the P2 residue has an R-configuration, are not hydrolysed by dipeptidyl peptidase IV.     

Structure-activity relationships of cyclic β-casomorphin-5 analogues

Cyclic analogues of the β-casein-derived opioid peptide β-casomorphin-5 (H-Tyr-Pro-Phe-Pro-Gly-OH) were prepared through substitution of the Pro² residue with various ,ω-diamino acid residues (lysine, ornithine, 2,4-diaminobutyric acid) and cyclization of the ω-amino group to the C-terminal carboxyl function. Compounds of this type, with D-configuration at the 2-position residue, showed high opioid receptor affinity with some preference for μ receptors over δ receptors, high potency in the guinea pig ileum assay and considerable activity in the mouse vas deferens assay. Configurational inversion at the 4-position in these cyclic analogues resulted in enhanced affinity for both μ and δ receptors, whereas N-methylation of the Phe³ residue produced a potency decrease.     

Selective protection of in vitro synthesized cDNA against nucleases by incorporation of phosphorothioate-analogues.

The conditions for the stepwise synthesis of single- (ss) and double-stranded (ds) cDNA using thio-analogues instead of dNTPs are described in this paper. RNA of paramyxovirus Sendai (strain 6/94) serves as template in these experiments. The increased resistance of this alpha S-modified cDNA against several nucleases, like S1-Nuclease, DNase I, Exonuclease III, snake venom Phosphodiesterase (PDE) and the combination of DNase I and PDE is demonstrated.     

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.