N-Terminal Sequence of Pig Brain Choline Acetyltransferase Purified by a Rapid Procedure

A procedure is reported that allows the purification and amino terminal sequencing of pig brain choline acetyltransferase. The enzyme (present in extremely low amounts in this tissue) is eluted together with its antibody from an affinity column by a mild pH shift and the resulting enzyme-antibody complex separated by gel electrophoresis. The band corresponding to the enzyme is electroeluted from the gel using volatile solutions allowing the direct determination of the amino acid composition and partial sequence. The first 11 residues are: Pro-Ile-Leu-Glu-Lys-Thr-Pro-Pro-Lys-Met-Ala.     

Modulation of differentiation markers in human choriocarcinoma cells by extracellular matrix: on the role of a three-dimensional matrix structure

Abstract. During spontaneous or chemically induced differentiation human choriocarcinoma cells express typical characteristics of the normal differentiating trophoblast: 1) increased production of peptide and steroid hormones (chorionic gonadotropin, placental lactogen, estrogens, progesterone); 2) increased activity of cellular alkaline phosphatase; 3) morphological transition from cytotrophoblast to syncytiotrophoblast-like cells; and 4) arrested cell proliferation. Since the extracellular matrix is known to control gene expression we have examined the effects of different substrates composed of matrix macromolecules on the differentiation of BeWo choriocarcinoma cells. Matrices tested were; fibronectin, laminin, collagens type I and type IV, the basement membrane-like complex matrix Matrigel, and a complex matrix extracted from human term placenta. Irrespective of the type of molecule(s), it was consistently found that, whenever the matrix molecules were presented as threedimensional structures (as opposed to protein coatings on tissue culture plastic) the response of affected differentiation markers monitored was highly pronounced. Morphology was changed from monolayers to rounded colonies, cell proliferation was reduced, and the secretion of chorionic gonadotropin was increased up to tenfold. Heterogeneous effects were observed on progesterone secretion and on the activity of cellular alkaline phosphatase. Cell adhesion to matrix molecules, however, did not depend on the structure of the matrix. This study demonstrates that gene expression in these tumor cells can be modified by extracellular matrix and highlights that not only the presence of effector molecules in the matrix but also the three-dimensional structure of the matrix is important for the induction of differentiation.     

Is the initiation of macronuclear DNA synthesis in Euplotes dependent on micronuclear functions?

To determine whether the micronucleus makes essential contributions during asexual reproduction, observations were made on cells of Euplotes octocarinatus from which the micronucleus had been removed with a micropipette. Most cells underwent one postenucleation division, then became arrested in macronuclear G1, slowed down in food uptake, developed macronuclear deformations, and finally died. Such cells could be rescued if a micronucleus was reimplanted before macronuclear deformations had developed. When provided with a new micronucleus, cells initiated macronuclear DNA synthesis about 12-16 h later. The data suggest that the micronucleus is involved in the control of the cell's transition from macronuclear G1 to S, and a model is proposed which postulates that in Euplotes macronuclear DNA synthesis is initiated when a micronucleus-encoded "initiator protein" has accumulated to a critical amount.     

Beobachtungen zur Lebensweise von Pycnogonum litorale (Ström) (Pantopoda)

Zusammenfassung Am Leitdamm des Jadebusens lebt Pycnogonum litorale im Lückensystem des Miesmuschelbesatzes. Dieser bietet mit hartem Untergrund, hoher Feuchtigkeit bei Niedrigwasser, genügend Actinien als Nahrung und guter Durchströmung bei gleichzeitigem Schutz vor Vertragung—offensichtlich günstige Lebensbedingungen für Pycnogonum litorale.Der Eiablage im Februar geht eine Reiterstellung des Männchens auf dem Weibchen von durchschnittlich 24 Tagen voraus. Unter künstlichen Kurztagbedingungen kann diese Reiterstellung auch außerhalb der Fortpflanzungsperiode eingenommen werden. Die Eier werden durch Rumpfbewegungen beider Partner zu den Ovigeren des Männchens bewegt. Bei 12°C schlüpfen die Larven etwa 41 bis 46 Tage nach der Eiablage aus, bei 19°C, im Sommer, schlüpften keine Larven.Im Jadebusen leben die Larven etwa 1/2 Jahn endoparasitisch in Hydrozoen. Die an die Metamorphose anschließende juvenile Phase, in der die Tiere frei leben, dauert ein knappes Jahr, die Reifehäutung erfolgt normalerweise im Sommer des zweiten Jahres, die Fortpflanzungsperiode etwa 6 Monate später, im Winter.

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Observations on the life biology of Pycnogonum litorale (Ström) (Pantopoda)

Summary Pycnogonum litorale lives in an interstitial system, of the mussel zone on the embankment of the Jadebusen. Hard substrate, high humidity at low tide, sufficient Metridium senile as food, and active currents together with protection from drifting, constitute favourable conditions for this pycnogonid.Prior to laying egg in February, the male remains in a riding position upon the female for approximately 24 days. Under artificial short-day conditions the riding position may also be assumed outside of the reproductive period. The eggs are transported to the ovigers of the male by trunk movements of both partners. At 12°C the larvae hatch about 41–46 days after egg-laying. No larvae hatched from eggs laid during summer at 19°C.The larvae live endoparasitically in Hydrozoa for about 1/2 year. Following metamorphosis, the freeliving juvenile phase lasts barely a year. The maturation moult normally takes place in the summer of the second year, the reproductive period beginning about 6 months later, in winter.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

The PIR-International Protein Sequence Database.

PIR-International is an association of macromolecular sequence data collection centers dedicated to fostering international cooperation as an essential element in the development of scientific databases. A major objective of PIR-International is to continue the development of the Protein Sequence Database as an essential public resource for protein sequence information. This paper briefly describes the architecture of the Protein Sequence Database and how it and associated data sets are distributed and can be accessed electronically.     

Cytotoxic effects of 6-mercaptopurine on the limb-bud blastemal cells of rat embryos.

On day 12 of pregnancy Wistar rats were each given a single ip injection of 5, 8, 16, 25, or 50 mg/kg 6-mercaptopurine. The embryos were removed 1, 2, 3, 5, 6, 10, 24, 48, or 72 h after injection or on day 20 and studied by light and electron microscopy. After 25 or 50 mg/kg all embryos showed no mineralization in the lower extremities. By electron microscopy condensation, shrinking, and fragmentation of cells in the limb bud blastema could be seen after 5 h. The fragments were phagocytosed and broken down by neighboring cells or remained in the extracellular space. After 25 or 50 mg/kg the damage was so extensive that the number of undamaged cells and of cells transforming into phagocytes was not sufficient to remove the debris or to compensate for the defect by mitotic activity. Epithelial cells, nerves, and blood vessels, show no morphological signs of damage. The "critical period" was the time cartilage just starts developing, i.e., when the blastema begins to differentiate.     

Stimulation of the diadinoxanthin cycle by UV-B radiation in the diatom Phaeodactylum tricornutum

In this study we show that the diadinoxanthin cycle in the diatom Phaeodactylum tricornutum is stimulated by mild UV-B radiation. High steady state concentrations of diatoxanthin established during a period of strong actinic illumination with white light (300 mol photons m-2 s-1 PAR) are further increased if weak UV-B (3 mol photons m-2 s-1) is additionally applied. Short term increases in the diatoxanthin concentration caused by UV-B strongly correlate with a stoichiometric decrease in diadinoxanthin. The UV-B dependent increase in diatoxanthin is correlated with a concommitant enhancement of non-photochemical quenching of chlorophyll fluorescence and a decrease in the quantum efficiency of oxygen evolution. This indicates that UV-B induced diatoxanthin functions in thermal energy dissipation. Possible scenarios for a stimulation of the diadinoxanthin cycle by UV-B are discussed.     

Conservation of protein-protein interactions - lessons from ascomycota

Interacting proteins from Saccharomyces cerevisiae are evolutionarily conserved and their likelihood of having an ortholog in other ascomycota species correlates with the number of interaction partners. Moreover, interacting proteins show a clear preference to be conserved as a pair, indicating that nature maintains selection pressure on the interaction links between proteins. The conservation of interacting protein pairs between different organisms does not exhibit any bias with respect to protein functional roles.     

Establishing a versatile fermentation and purification procedure for human proteins expressed in the yeasts Saccharomyces cerevisiae and Pichia pastoris for structural genomics

We describe the introduction of the yeasts Saccharomyces cerevisiae and Pichia pastoris as eukaryotic hosts for the routine production of recombinant proteins for a structural genomics initiative. We have previously shown that human cDNAs can be efficiently expressed in both hosts using high throughput procedures. Expression clones derived from these screening procedures were grown in bioreactors and the over-expressed human proteins were purified, resulting in obtaining significant amounts suitable for structural analysis. We have also developed and optimized protocols enabling a high throughput, low cost fermentation and purification strategy for recombinant proteins for both S. cerevisiae and P. pastoris on a scale of 5 to 10 mg. Both batch and fed batch fermentation methods were applied to S. cerevisiae. The fed batch fermentations yielded a higher biomass production in all the strains as well as a higher productivity for some of the proteins. We carried out only fed batch fermentations on P. pastoris strains. Biomass was produced by cultivation on glycerol, followed by feeding methanol as carbon source to induce protein expression. The recombinant proteins were expressed as fusion proteins that include a N-terminal His-tag and a C-terminal Strep-tag. They were then purified by a two-step chromatographic procedure using metal-affinity chromatography and StrepTactin-affinity chromatography. This was followed by gel filtration for further purification and for buffer exchange. This three-step purification procedure is necessary to obtain highly purified proteins from yeast. The purified proteins have successfully been subjected to crystallization and biophysical analysis.