Verteilung und Aufteilung der DNS bei einigen Cyanophyceen,festgestellt durch ihre DAPI-Fluoreszenz

The DNA of Blue-Green Algae may be specifically and most efficiently elucidated by the combined use of the fluorochrome DAPI and DNAse treatment. InChroococcus turgidus andSynechococcus aeruginosus it forms a network which usually is positioned at the periphery of the centroplasm. At the beginning of cell division it seems to be invaginated by the ingrowing cell wall. Later it must be distributed by other means because two equal parts are connected by a number of fine and straight DNA threads. InOscillatoria limosa usually all parts of the centroplasm are interspersed with the DNA network. In surface view, the DNA occasionally seems to consist of a number of independant rods, but in reality these are connected to the general network. Division apparantly occurs by ingrowth of the crosswall and usually results in equal daughter nuclear equivalents although occasionally they are unequal. Four strains ofMicrocoleus vaginatus were found to have nuclear equivalents of the same general appearance as inOscillatoria limosa.

     

Elektronenmikroskopischer nachweis saurer mucopolysaccharide in den flaschenzellen der Xenopus-niere

Zusammenfassung Flaschenzellen, eigenartige flaschenförmige Epithelzellen im Verbindungsstück der Xenopusniere, enthalten ein stark entwickeltes intrazelluläres Kanalsystem, das mit sauren Mucopolysacchariden gefüllt ist. Bei Anwendung der Methode mit kolloidalem Ferriammoniumglyzerat (pH 1,6) erweisen sich das Material im Kanalsystem, in den Vesikeln in der Nachbarschaft der intrazellulären Kanäle, einige Golgi-Zisternen und Autophagosomen als positiv. Ein dichter Belag von positivem Material wurde auch an der luminalen Oberfläche der Epithelzellen des Verbindungsstückes gefunden.In Fröschen, die monatelang in Salzwasser gehalten wurden, war das intrazelluläre Kanalsystem zurückgebildet. Sein reduzierter Inhalt zeigte eine verminderte Reaktion und im Zytoplasma waren nur ausnahmsweise positiv reagierende Vesikel und Golgi-Zisternen zu beobachten. Wenn die Tiere wieder in Süßwasser gesetzt werden, kommt es zu einer Anhäufung positiver Vesikel und Golgiapparate und einer Zunahme des Materials im Kanalsystem.

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Ultrastructuml localization of acid mucopolysaccharides in the so-called flask cells of the Xenopus nephron

Summary Flask cells (Flaschenzellen), peculiar bottle-shaped epithelial cells in the intermediate segment of the Xenopus nephron, contain a highly developed intracellular channel system filled with acid mucopolysaccharides. The cells were studied with the colloidal ferriammoniumglycerate method at pH 1,6 at the electron microscopic level. The material inside the intracellular channels, cytoplasmic vesicles in the neighbourhood of the latters, several cisternae of the Golgi apparatus and some autophagosomes were found to be positive. A thick coat of positive material was observed also on the luminal surface of the epithelial cells in the intermediate segment.In frogs living in salt water for 2 months, the intracellular channels are strongly under-developed, their reduced content shows a weak reaction; positive vesicles and Golgi cisternae occur exceptionally. If the animals were returned to fresh water, an increase of the reactive vesicles and Golgi cisternae as well as the contents of the channels could be observed.

Für die Überlassung des Themas sind wir Herrn Professor L. Spannhof (Rostock) zu Dank verpflichtet.     

The effect of ethanol on cell wall antigens ofSaccharomyces cerevisiae and specific isolation of high ethanol producing strains of this yeast,making use of a serological technique

Generally, natural isolates of high ethanol producingSaccharomyces cerevisiae obtained by screening are used in alcoholic industries. The methods involved in their isolation and identification are elaborate. Antigenic analysis using antibodies raised against wholeSaccharomyces cells indicated species specificity of cell wall surface thermostable antigens. By affinity purification, the specific antibodies could be obtained and used for specific isolation ofS. cerevisiae. Antigenic studies using antibodies raised against isolated cell walls of fermentatively grownS. cerevisiae indicated the occurrence of thermolabile antigens common toSaccharomyces species. Higher concentrations of these antigens could be detected in thoseS. cerevisiae that had the ability for high ethanol production. The concentrations of these cell wall common antigens increased with increasing culture age and ethanol accumulation in culture broths. In younger yeast cells, the concentration could be increased by growing the cells in a medium containing added ethanol. Using dilutions of cross absorbed antibody specific for common antigens and Ouchterlony test, high ethanol producingS. cerevisiae could be identified.     

Mechanics of circadian pulvini movements in Phaseolus coccineus L.

The circadian movement of the lamina of primary leaves of Phaseolus coccineus L. is mediated by antagonistic changes in the length of the extensor and flexor cells of the laminar pulvinus. The cortex of the pulvinus is a concentric structure composed of hexagonal disc-like cells, arranged in longitudinal rows around the central stele. Observations with polarization optics indicate that the cellulose microfibrils are oriented in a hoop-like fashion in the longitudinal walls of the motor cells. This micellation is the structural basis of the anisotropic properties of the cells: tangential sections of the extensor and flexor placed in hypotonic mannitol solutions showed changes only in length. As a consequence a linear correlation between length and volume was found in these sections. Based on the relationship between the water potential (which is changed by different concentrations of mannitol) and the relative volume of the sections and on the osmotic pressure at 50% incipient plasmolysis, osmotic diagrams were constructed for extensor and flexor tissues (cut during night position of the pulvinus). The bulk moduli of extensibility, , were estimated from these diagrams. Under physiological conditions the values were rather low (in extensor tissue below 10 bar, in flexor tissue between 10 to 15 bar), indicating a high extensibility of the longitudinal walls of the motor cells. They are strongly dependent on the turgor pressure at the limits of the physiological pressure range.In well-watered plants, the water potentials of the extensor and flexor tissues were surprisingly low,-12 bar and-8 bar, respectively. This means that the cells in situ are by no means fully turgid. On the contrary, the cell volume in situ is similar to the volume at the point of incipient plasmolysis: the cell volumes of extensor and flexor cells in situ were only 1.01 times and 1.1 times larger, respectively, than at the point of incipient plasmolysis, whereas at full turgidity (cells in water) the corresponding factors were 1.8 and 1.5. It is suggested that the high elasticity of the longitudinal walls, the anisotropy of the cell walls, and the low water potential of the sections which is correlated with slightly stretched cell walls in situ, are favourable and effective for converting osmotic work in changes in length of the pulvinus cells, and thus for the up and down movement of the leaf.Symbols volumetric elastic modulus - _i instantaneous volumetric elastic modulus - _i stationary volumetric elastic modulus - weight-averaged stationary bulk modulus of extensibility - ₀ osmotic pressure of the vacuole of a cell at the point of incipient plasmolysis - weight-averaged osmotic pressure of the vacuoles of the tissue at 50% incipient plasmolysis - water potential     

Relationship between microtubule orientation and patterns of cell expansion in developing cortical cells of Lemna minor roots

In actively growing cortical cells in the elongation zone of Lemna minor L. roots, both longitudinal (radial and tangential) and transverse walls expand in both length and width. The longitudinal walls of the three types of cortical cells in the root (i.e. outer, middle and inner) showed the largest expansion in the longitudinal axis. In contrast, the inner cortical cells exhibited the least expansion in width, whereas the middle cortical cells displayed the largest expansion in width. Thus, the profiles of the expansion of longitudinal walls were characteristic for the three types of cortical cells. In this study, both the orientation of cortical microtubule (MT) arrays and their dynamic reorientation, and the density of cortical MTs, were documented and correlated to the patterns of cell wall expansion. Significantly, transverse arrays of cortical MTs were most prominent in the radial walls of the inner cortical cells, and least so in those of the middle cortical cells. Toward the base of roots, beyond the elongation zone, the orientation of cortical MTs shifted continuously from transverse to oblique and then to longitudinal. In this case, the rate of shift in the orientation of cortical MTs along the root axis was appreciably faster in the middle cortical cells than in the other two types of cortical cells. Interestingly, the continuous change in cortical MT orientation was not confirmed in the transverse walls which showed much smaller two-dimensional expansion than the radial walls. Additionally, the presence of fragmented or shortened cortical MTs rapidly increased concomitantly with the decrease of transversely oriented cortical MTs. This relationship was especially prominent in the transverse walls of the inner cortical cells, which displayed the least expansion among the three types of cortical cells investigated. In the root elongation zone, the density of cortical MTs in the inner cortical cells was about three times higher than that in the other two cortical cell types. These results indicate that in the early stage of cell expansion, the orientation of cortical MTs determines a preferential direction of cell expansion and both the shifting orientation and density of cortical MTs affect the magnitude of expansion in width of the cell wall.     

Characterization of microstructure and cell wall components of Arabidopsis thaliana overexpressing cyclin-dependent kinase inhibitor 2

Overexpression of a cyclin-dependent kinase inhibitor (KRP2) caused changes in the general morphology in the leaves of Arabidopsis thaliana. The wild type plant had obovate leaves with entire margins whereas the transgenic line had leaves with denticulate margins. The epidermal cells and stomata of the adult transgenic leaves were significantly larger than those of the wild-type plants and the number of stomata was in proportion to the number of epidermal cells. No apparent differences in thickness and structure of cell walls of the mesophyll cells between the two samples were observed. The smaller amount of cell wall material in the transgenic leaves caused by the larger cell size was also apparent in the lower dry weight of the transgenic leaves. The chemical analysis revealed the main differences to be in pectin and neutral sugar contents, and especially in the amounts of glucose, all being higher in the leaves of the KRP2 transgenic plants. p-Coumaric acid content varied more in the transgenic leaf material than in the control one reflecting possibly fewer cross-links in the cell walls of transgenic plants.     

Isolation of the promoter of a root cap expressed pectinmethylesterase gene from Pisum sativum L. (rcpme1) and its use in the study of gene activity

A genomic clone of a pea pectinmethylesterase encoding gene, rcpme1, was isolated; the promoter region was found to include regions of homology to phenylalanine ammonia lyase (PAL) and nodulin gene promoters. Agrobacterium rhizogenes mediated hairy roots were used for rcpme1 expression and functional analysis in pea. Patterns of rcpme1 expression in cultured hairy roots, measured using uidA encoding -glucuronidase (GUS) as a reporter gene, were distinct from patterns which occur in normal pea roots. No reporter gene expression occurred in transgenic Arabidopsis thaliana, whose roots do not produce border cells. Border cell number from transgenic hairy roots expressing rcpme1 anti-sense mRNA under the control of its 2.75 kb 5 flanking sequence was reduced by > 50%. Nodulation genes of Rhizobium leguminosarum were used as a marker to document that roots with reduced production of border cells and other root cap exudates have a corresponding reduction in levels of biologically active signal molecules. Direct measurements were used to confirm that most of the exudate harvested from young, unwounded roots of normal pea plants is derived from the root tip region where rcpme1 is expressed. The potential application of the rcpme1 gene as a molecular marker for root exudate production is discussed.     

Expression of a foreign eukaryotic gene in Saccharomyces cerevisiae: β-galactosidase from Kluyveromyces lactis

Three recombinant DNA vectors carrying the β-galactosidase structural gene, LAC4, from the yeast Kluyveromyces lactis were constructed and transformed into Saccharomyces cerevisiae. All transformants expressed the β-galactosidase activity of LAC4. However, the level of enzyme activity varied, being highest in cells transformed with vectors which are maintained as multicopy plasmids and lowest in cells transformed with a vector which integrates into chromosomes. Enzyme levels probably reflect gene dosage. LAC4 is very stable when integrated into a chromosome, but unstable when carried on a plasmid. Therefore, stability is a property of the recombinant vector rather than of LAC4, LAC4-coded β-galactosidase synthesized in either S. cerevisiae or in K. lactis is the same as judged by two-dimensional polyacrylamide gel electrophoresis. However, S. cerevisiae transformed with     

Exocellular (glyco)proteins of Lupinus albus plants and suspension-cultured cells

Plant species from genus Lupinus are among the oldest known legumes, and various aspects of their biology are considerably different from those commonly observed within Leguminosae. To study this issue in more detail, a suspension culture of Lupinus albus cells was developed, and the glycosylation patterns of exocellular proteins analysed. N-linked oligosaccharide side-chains were detected with two lectins: concanavalin A (ConA) and wheat germ agglutinin (WGA) used with respective anti-lectin antibodies, while O-linked arabinosylated side-chains of (hydroxy)proline-rich glycoproteins were identified with anti-(42 kDa French bean chitin-binding protein) antibodies. The obtained data were compared with analogous ones for exocellular (glyco)proteins from suspension-cultured Phaseolus vulgaris cells and from various tissues of L. albus plants. Major species-specific differences between exocellular (glyco)proteins from lupin and bean cells were identified. Similarly, developmentally regulated glycosylation changes following transition from organised plant tissue to dedifferentiated suspension-cultured lupin cells were detected and analysed.     

Cyanide-induced death of cells in plant leaves

Destruction of guard cell nuclei in epidermis isolated from leaves of pea, maize, sunflower, and haricot bean, as well as destruction of cell nuclei in leaves of the aquatic plants waterweed and eelgrass were induced by cyanide. Destruction of nuclei was strengthened by illumination, prevented by the antioxidant alpha-tocopherol and an electron acceptor N,N,N ,N -tetramethyl-p-phenylenediamine, and removed by quinacrine. Photosynthetic O2 evolution by the leaf slices of a C3 plant (pea), or a C4 plant (maize) was inhibited by CN- inactivating ribulose-1,5-bisphosphate carboxylase, and was renewed by subsequent addition of the electron acceptor p-benzoquinone.     

Cell walls as reservoirs of potassium ions for reversible volume changes of pulvinar motor cells during rhythmic leaf movements

The laminar pulvinus of primary leaves of Phaseolus coccineus L. was investigated with respect to the total K⁺ content, the apoplastic K⁺ content, and the water potential of extensor and flexor sections in relation to the leaf positions in a circadian leaf-movement cycle, as well as the cation-exchange properties of isolated extensor- and flexor-cell walls. Turgid tissue showed a high total but low apoplastic K⁺ content, shrunken tissue a low total but high apoplastic K⁺ content. Thus, part of the K⁺ transported into and out of the swelling or shrinking protoplasts is shuttled between the protoplasts and the surrounding walls, another part between different regions of the pulvinus. The K⁺ fraction shuttled between protoplasts and walls was found to be 30–40% of the total transported K⁺ fraction. Furthermore, 15–20% of the total K⁺ content of the tissue is located in the apoplast when the apoplastic reservoir is filled, 5–10% when the apoplastic reservoir is depleted. The ion-exchange properties of walls of extensor and flexor cells appear identical in situ and in isolated preparations. The walls behave as cation exchangers of hhe weak-acid type with a strong dependence of the activity of fixed negative charges as well as of the K⁺-storing capacity on pH and [K⁺] of the equilibration solution. The high apoplastic K⁺ contents of freshly cut tissues reflect the cation-storing capacity of the isolated walls. We suggest that K⁺ ions of the Donnan free space are used for the reversible volume changes (mediating the leaf movement) mainly by an electrogenic proton pump which changes the pH and-or the [K⁺] in the water free space of the apoplast.Abbreviations and symbols DFS Donnan free space - DW dry weight - pK negative logarithm of the equilibrium constant K of the acidic group - WFS water free space - water potential; Indices - cw cell wall - t tissue     

Tachykinin NK-1 receptor probed with constrained analogues of substance P

The action of rotameric probes introduced either in position 7 or 8 in the sequence of substance P (SP) was investigated, i.e. -tetrahydroisoquinoleic acid (Tic), -fluorenylglycine (Flg), -diphenylalanine (Dip), the diastereoisomers of -1-indanylglycine (Ing) and -benz[ƒ]indanylglycine (Bfi), the Z- and E-isomers of dehydrophenylalanine and dehydronaphthylalanine (Δ^ZPhe, Δ^EPhe, Δ^ZNal, Δ^ENal) and (Dmp). The aim of this study was the topographical characterization of the binding subsites of human NK-1 receptor expressed in CHO cells, especially the S₇ and S₈ subsites, corresponding to residues Phe⁷ and Phe⁸ of substance P. According to the binding potencies of these substituted-SP analogues, the S₇ binding subsite is smaller than the S₈ subsite: the S₇ subsite accepts only one aromatic nucleus, while the S₈ can accommodate three coplanar nuclei altogether. These findings are compatible with the idea that the S₈ binding subsite may reside in the extracellular loops of the hNK-1 receptor. NK-1 agonists bind to human NK-1 receptor and activate the production of both inositol phosphates and cyclic AMP. As already quoted for septide, [pGlu⁶, Pro⁹]SP(6–11), discrepancies are observed between affinity (K_i) and activity (EC₅₀) values for IPs production. While a weak correlation between K_i and EC₅₀ values for IPs production could be found (r = 0.70), an excellent correlation could be demonstrated between their affinities (K_i) and their potencies (EC₅₀) for cAMP production (r = 0.97). The high potency (EC₅₀) observed for ‘septide-like’ molecules on PI hydrolysis, compared to their affinity is not an artefact related to the high level of NK-1 receptors expressed on CHO cells since a good correlation was found between EC₅₀ values obtained for PI hydrolysis and those measured for spasmogenic activity in guinea pig ileum bioassay (r = 0.94).

According to the binding potencies of constrained analogues of phenylalanine, the S₇ binding subsite of human NK-1 receptor is small, whereas the S₈, which can accommodate three coplanar nuclei, might probably reside in the extracellular loop. The discrepancies observed between affinity (K_i) and activity (EC₅₀) values for IPs production are not an artefact of CHO cells since a good correlation was found between EC₅₀ for PI hydrolysis and those measured in guinea pig ileum bioassay.     

Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by PhiC31 integrase

The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3ζ/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of FcγRII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.