A bioreactor model system specifically designed for Tetrahymena growth and cholesterol removal from milk.

This work describes the configuration and operation of a bioreactor system especially designed for Tetrahymena cultivation and its use for milk improvement, particularly cholesterol elimination by the action of this cell. An advantage of the proposed method is the re-use of the growth medium; thus, the medium is used twice to provide two batches of Tetrahymena biomass without the need of further inoculation. This makes the procedure of producing the cell biomass faster and more economical. Cells are concentrated in the culture vessels by sedimentation at room temperature and then transferred to milk suspensions, where they can further grow for at least one generation with the benefit of reducing steeply cholesterol level. Milk treated according to this process is separated from the biomass by centrifugation. Under these conditions, less than 5% of the cells remain in the milk, and cholesterol elimination amounts to 75 +/- 10% of that initially present. No changes in sensorial properties of the milk, such as clotting or butyric odor, were observed as a result of this treatment. In addition, the bioreactor allows the aseptic recovery of the spent growth medium, which contains diverse enzymes of interest, and the cell pellets, to exploit particular lipids like phosphonolipids, abundant poly-unsaturated fatty acids and co-enzyme Q(8).

The use of Tetrahymena thermophila mutant cell line for removal of cholesterol from milk.

The nonpathogenic ciliate Tetrahymena thermophila converts cholesterol from foodstuffs into provitamin D compounds in high yields. However, prolonged incubation with wild-type strain CU-399 at high densities results in a final deterioration of milk properties, possibly as a result of secreted hydrolases. Here we attempted to solve this problem using MS-1 Tetrahymena strain, a stable mutant with a low rate of hydrolase secretion. Densities of to 2 x 10( 6 ) cells/ml can be incubated for up to 5 h in milk, without any clotting or change in appearance. Moreover, centrifugation of this suspension eliminates most of the cells, and results in an about 75% +/- 10 (n = 10) decrease of the initial cholesterol. Sterols are recovered in the cell pellets, which show that Tetrahymena is able to avidly capture them from the medium. Therefore, this mutant strain is optimal for milk cholesterol depletion, avoiding unfavorable sensory alterations.

Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila.

We have described a procedure for the isolation of mutants of Tetrahymena thermophila with hypersecretion of phospholipase A1 (PLA1). Using random chemical mutagenesis, uniparental cytogamy, genetic crossing and a new, fast and effective screening procedure, four PLA1-hypersecretory mutants were isolated. The screening procedure is based on the formation of a halo appearing around cylindrical holes in a lecithin-containing agar plate filled with cell-free supernatants. About 3,940 clones were tested with this procedure in primary screening for hypersecretory features, of which 60 putative hypersecretory mutants were isolated, subcloned and tested in a secondary screening. Of these, four selected mutants showed 1.8-2.2 more PLA1 activity in the cell-free supernatants compared to the wild-type strain CU 438.1. Hypersecretion was only observable for PLA1; no increased activity for two other lysosomal enzymes could be detected. These hypersecretory mutants of T. thermophila can be very useful for increasing the yield of PLA1 in fermentation processes. This is particularly relevant because, in contrast to other phospholipases, PLA1 is not available on the commercial market for fine chemicals and little is known about the role of PLA1 in cell signaling and metabolism.

Intra- and intercellular communication systems in ciliates.

Intracellular signaling and cell-cell interactions are basic features of living organisms. Ciliated protozoa show complex mechanisms of intracellular signaling, as is demonstrated for the phagosomal pathway. Although unicellular, ciliates also communicate with other cells, for example, with invading or symbiotic micro-organisms, some of which are dwelling in the nuclei. In predator-prey interactions chemical signals (kairomones) released by certain predators induce defensive morphological or behavioral changes in the prey ciliates. In intercellular communication sensu strictu ciliate cells communicate with each other, for example, in sexual propagation. A variety of sexual signals have been found to function in preconjugant interaction. Many phenomena of cellular communication in ciliates appear to be similar to those found in multicellular organisms.

Biochemical analysis of membrane proteins from an early maturation stage of phagosomes.

We used an improved technique for pulse-chase labeling of phagosomes using custom-made magnetic microparticles. With the help of a permanent magnet we purified both newly formed, nascent and early matured (i.e., 5-min-old) condensed phagosomes in high amounts. The protein patterns of membrane proteins of newly formed phagosomes and 5-min-old condensed ones were compared by two-dimensional (2-D) electrophoresis. The protein patterns allowed the detection of protein spots that changed in abundance between these two stages. Three protein spots abundant in condensed phagosomes only and one spot well-stained in both stages were collected from ten preparative Coomassie brilliant blue-stained 2-D gels. Following microdigestion, selected purified oligopeptides were sequenced by Edman degradation. While the oligopeptide sequences of proteins from two spots showed high homology to an already sequenced 25 kDa calcium binding protein, the other two showed no significant homology to protein sequences available in sequence databases. Presently polymerase chain reaction (PCR) and cloning experiments are set up to reveal the cDNAs of these proteins in order to study their function by knock-out and gene replacement experiments.

A method for the preparation of Tetrahymena thermophila phospholipase A1 suitable for large-scale production.

A rapid and economical method for the purification of phospholipase A1 (PLA1) from the extracellular medium of the ciliate Tetrahymena thermophila is presented. Essentially, the procedure, here designated as purification by selective interaction (PSI), entails the incubation of media containing PLA1 with liposomes made of soy bean phospholipids. The PLA1-lipid complexes are precipitated by the addition of CaCl2 and collected by centrifugation. Elution of the PLA1 is effected by treating the complexes with 40% dimethylformamide, a reversible inhibitor of this enzyme, which is easily removed by dialysis. In combination with DEAE cellulose ion exchange chromatography, PSI yielded homogeneous PLA1 preparations with a 14% recovery and a 416-fold increase in specific activity. This procedure, which can be completed within 1 day, may prove useful for the isolation of phospholipases from other sources. This practical method for the purification of a microbial PLA1 opens the way to large-scale production of these types of enzyme, which are not as yet commercially available.

Maturation of phagosomes is accompanied by specific patterns of small GTPases.

In this study we purified phagosomes of the ciliated protozoan Tetrahymena thermophila to analyze aspects of the maturation pathway of phagocytotic vesicles. Phagosomes were labeled with magnetic microparticles and then purified in high amounts with the help of a permanent magnet. By combining a pulse-chase labeling protocol with the magnetic separation procedure we were able to isolate phagosomes of defined ages, which represent distinct stages of their maturation pathway. GTP-overlay assays showed that a set of small GTPases of the ras superfamily is associated with these phagosomes. Phagosomes isolated at different stages of maturation revealed a change in the pattern of the small GTPases. Some small GTPases identified by the GTPase overlay assays could be aligned to India ink stained protein spots in two-dimensional gels of isolated phagosomes. The results presented here are a first step to identify the members of small GTPases associated with phagosomes during their maturation pathway. Microsequencing of pooled polypeptides by mass-spectrometric techniques will identify the primary structure of these small GTPases.

[Current aspects of diagnosis and therapy of appendicitis in childhood]. / Aktuelle Aspekte der Diagnostik und Therapie der Appendizitis im Kindesalter.

627 appendectomies in childhood, performed since 1990 to 1996 at the Clinic of Paediatric surgery of the Neubrandenburg Hospital, were analysed. It's a retrospective study about aspects of diagnosis, differential diagnosis and surgical treatment.

Tetrahymena: a model for growth, cell cycle and nutritional studies, with biotechnological potential.

Tetrahymena has been used as a model cell system in many studies of morphogenesis, conjugation, gene mapping, cell division and growth kinetics. In this article, we consider some advances which have resulted from the successful development of a chemically defined medium (CDM), and how subsequent work has extended the contribution that this organism has made to our understanding of different aspects of growth, nutrition, cell cycle control, cytokinesis and intercellular signalling. Finally, we discuss the considerable potential that has arisen for the biotechnological exploitation of this big and rapidly growing eukaryotic cell.

Processing and secretion of lysosomal acid alpha-glucosidase in Tetrahymena wild type and secretion-deficient mutant cells.

The proteolytic processing and secretion of a lysosomal enzyme, acid alpha-glucosidase, was studied by pulse-chase labeling with [35S]methionine in Tetrahymena thermophila CU-399 cells treated with ammonium chloride. This cell secreted a large amount of acid alpha-glucosidase into the cultured medium during starvation. The secretion was found to be repressed by addition of ammonium chloride (NH4Cl). Acid alpha-glucosidase was produced as a precursor form (108 kDa) and then processed to a mature polypeptide (105 kDa) within 60 min. This mature enzyme was secreted into the media within 2-3 h after chase, whereas the precursor form was not secreted by either control cells or NH4Cl-treated cells. NH4Cl did not affect the processing of the precursor acid alpha-glucosidase. Processing profile of this enzyme was apparently indistinguishable from that of the mutant MS-1 defective in lysosomal enzyme secretion. Furthermore, the purified extracellular (CU-399) and intracellular (MS-1) acid alpha-glucosidases were the same in molecular mass (105 kDa) and enzymatic properties. They contained no mannose 6-phosphate residues in N-linked oligosaccharides. These results suggested that unlike mammalian cells, Tetrahymena acid alpha-glucosidase may be transferred to lysosomes by a mannose 6-phosphate receptor-independent mechanism, and also that low pH was not essential for the proteolytic processing of precursor polypeptide.

Three pools of lysosomal enzymes in Tetrahymena thermophila.

Secretion of lysosomal enzymes into the extracellular surroundings has been observed in many eukaryotic cells. We studied the activity of lysosomal enzymes in different subcellular fractions of Tetrahymena thermophila to get more insight into this general phenomenon. By density gradient centrifugation a light and a dense fraction of lysosomal particles were found. Electron microscopy revealed that the light fraction mainly consists of cell surface membranes. By immunostaining a lysosomal enzyme (beta-hexosaminidase) was detected on the plasma membrane. The Triton X-114 assay showed that the light fraction as well as purified cilia (an enriched source of plasma membrane) contain lysosomal enzymes predominantly covalently bound to the membrane. The dense fraction contains both membrane-bound and soluble forms of lysosomal enzymes. By labeling phagosomes/phagolysosomes with magnetic particles the dense fraction can be subdivided into two lysosomal vesicle populations: phagolysosomes and a further population of lysosomal vesicles which can not be labeled. The relationship between membrane-bound and soluble enzyme forms in phagolysosomes and this unlabeled vesicle population is different: In phagolysosomes 80% of the acid phosphatase and 20% of the beta-hexosaminidase are membrane-bound, whereas in the unlabeled vesicles 42% of the acid phosphatase and 8% of the beta-hexosaminidase are bound to the membrane. Furthermore, we present results suggesting that the unlabeled vesicle population of the dense fraction is the source of secreted lysosomal enzymes. A working model summarizing our present knowledge about the connection of the three pools of lysosomal enzymes in Tetrahymena is presented.

The Golgi apparatus of Tetrahymena thermophila.

Electron microscopic investigations reveal that the Golgi apparatus of Tetrahymena thermophila consists of numerous tiny dictyosomes, each consisting of one or two cisternae. The dictyosomes are localized predominantly in the cell cortex closely associated with the mitochondria, arranged in meridians alternating with the ciliary meridians. We estimated about 300-400 of these dictyosomes in the periphery of a cell, a value corresponding to the number of somatic cilia per cell. Cytochemical assays of thiamine pyrophosphatase and acid phosphatase, both marker enzymes of trans Golgi cisternae, resulted in deposits of lead or cerium phosphate in the outermost cisternae of the dictyosomes. In addition, cisternae located at the bases of the basal body/parasomal sac arrangements are stained. This indicates that these cisternae may belong to the Golgi apparatus of the cell.

Effects of immobilization on growth, morphology, and DNA content of the ciliated protozoon Tetrahymena thermophila.

Morphological and physiological properties of Tetrahymena thermophila immobilized by encapsulation in calcium-alginate hollow spheres were found to be substantially different from those of suspended cells. Immobilized T. thermophila reached lengths of 70-100 microns, whereas the average cell of suspension cultures was about 40 microns long. Suspended cells appeared typically pear-shaped while immobilized cells developed a proboscis-like anterior end. Contrary to suspended T. thermophila, encapsulated cells were functionally deficient in phagocytosis although developing an oral apparatus. The diameter of the macronucleus of immobilized cells was about two times larger than the macronucleus of suspended cells and contained twice as much DNA, while the DNA content of the micronucleus remained unchanged. High cell density fermentations of suspended cells indicated that the alterations observed in immobilized cells were not due to close physical contacts between the cells.

Continuous high-cell-density fermentation of the ciliated protozoon Tetrahymena in a perfused bioreactor.

An efficient method of growing the protozoon Tetrahymena to high cell densities in a 2-1 bioreactor is described. The first phase of the fermentation is a batch phase with minimum generation times (1.4 h). During the next phase medium is exchanged continuously using a perfusion module based on microporous hollow fibres while cell are retained. Compared to standard batch fermentations of this organism 30- to 40-fold higher cell concentrations and dry weights were achieved routinely. A maximum cell concentration of 2.2 x 10(7) cells/ml and a dry weight of 54 g/l have been obtained. As estimated from isocitrate dehydrogenase activity in the culture medium, no cell damage occurred even at high agitation rates. In addition, the cells remained viable for several weeks. Temporal limitation of the process was due to a decrease in the perfusion rate caused by blocking of the membranes. By X-ray microprobe analysis calcium phosphate depositions were detected in the pores of the clogged hollow-fibre membranes. However, even a T. pyriformis strain possessing mucocysts, dense core secretory organelles that may lead to early membrane clogging, was cultivated successfully for 3 weeks. Additionally, the consumption of nutrient protein and carbohydrates during fermentation was investigated and the effect of different perfusion rates and of glucose was studied in order to increase the efficacy of the system.

Differential increase in activity of acid phosphatase induced by phosphate starvation in Tetrahymena.

We have studied the effects of phosphate starvation on the levels and distributions of activities of acid phosphatase and beta-hexosaminidase in cultures of Tetrahymena thermophila. The cells were grown in synthetic nutrient medium and refed every day with fresh medium. After 4 days of growth in the complete medium, the cultures were divided into two portions. One received complete medium and the other phosphate-free, but otherwise complete, medium. Population densities and activities of acid phosphatase and beta-hexosaminidase in cells plus medium and in cell-free samples were determined in aliquots removed every day before medium replacement. In cultures having complete medium the enzyme levels remained fairly constant; in the phosphate-starved cultures both total and extracellular activities of acid phosphatase increased sixfold. beta-Hexosaminidase levels remained essentially unaltered in both cases. These results indicate that phosphate starvation can induce differential increase in acid phosphatase activity in cultures of Tetrahymena. Somewhat less than 50% of the total activities of both enzymes are found in the cell-free extracellular fluid at any time.

Genetic characterization of the secretory mutant MS-1 of Tetrahymena thermophila: vacuolarization and block in secretion of lysosomal hydrolases are caused by a single gene mutation.

The genetics and phenotypic features (light and electron microscopy) of a secretory mutant, MS-1 of Tetrahymena thermophila blocked in secretion of lysosomal acid hydrolases have been analyzed. Although blocked constitutively in secretion, MS-1 contains active lysosomal hydrolases in amounts equivalent to the wild type. The 3:1 segregation in F-2 in sib crosses and the 1:1 segregation in test crosses indicate that the block in secretion of lysosomal hydrolases is controlled by a recessive single gene locus termed sec. The sec allele of MS-1 proved also to be responsible for the highly vacuolarized phenotype the mutant developed when it was transferred from nutrient medium into buffers of low ionic strength. Deletion mapping by crossing MS-1 with nullisomic strains, all secreting lysosomal hydrolases at wild-type rates, was performed. The sec phenotype was expressed in monosomic-4 progeny only, indicating that the sec allele is located on chromosome 4 of T. thermophila.

Lysosomal enzymes produced by immobilized Tetrahymena thermophila.

The ciliated protozoon Tetrahymena thermophila was immobilized for production of secreted lysosomal enzymes in two ways. Cells entrapped in solid Ca-alginate spheres survived but were unable to grow and multiply. However, when encapsulated in hollow Ca-alginate spheres Tetrahymena multiplied well, reaching 0.9 x 10(7) cells/ml. These immobilized cells secreted large amounts of lysosomal enzymes when the medium was changed daily. This system was transferred to a reactor scale using a conical bubble column reactor for semicontinuous cultivation of the encapsulated cells. Under these conditions alpha-glucosidase, beta-glucosidase, beta-hexosaminidase and acid phosphatase were produced for at least 4 weeks. The hollow spheres were stable for 3 months and contained living and secreting Tetrahymena cells during this time. Immobilized T. thermophila cells can thus serve as a good source for production of commercially interesting enzymes.

The role of secreted acid hydrolases in the utilization of complex nutrients byTetrahymena.

In an attempt to extend our knowledge of the biology of feeding of the ciliateTetrahymena thermophila, this organism was grown axenically on complex organic material. The nutrient substrate was based on autoclaved wheat grains and adjusted to either pH 5.5 or 7.5. In wild type cultures the cells grew and multiplied only under acidic conditions. In cultures of a mutant cell line blocked in the secretion of acid hydrolases the cells did not grow at either pH value. Thus released acid hydrolases may play a key role in the utilization of complex nutrients in combination with uptake of small organic molecules. Mechanisms in the feeding biology ofTetrahymena thermophila andParamecium tetraurelia are compared.