Fusion of cultured dog kidney (MDCK) cells: I. Technique,fate of plasma membranes and of cell nuclei

Summary The evaluation of the intracellular signal train and its regulatory function in controlling transepithelial transport with electrophysiological methods often requires intracellular measurements with microelectrodes. However, multiple impalements in epithelial cells are hampered by the small size of the cells. In an attempt to avoid these problems we fused cells of an established cell line, Madin Darby canine kidney cells, originally derived from dog kidney, to giant cells by applying a modified polyethylene glycol method. During trypsin-induced detachment from the ground of the petri dish, individual cells grown in a monolayer incorporate volume and mainly lose basolateral plasma membrane by extrusion. By isovolumetric cell-to-cell fusion, spherical giant cells are formed within 2 hr. During this process a major part of the individual cell plasma membranes is internalized. Over three weeks following cell plasma membrane fusion degradation of single cell nuclei and cell nuclear fusion occurs. We conclude that this experimental approach opens the possibility to investigate ion transport of epithelia in culture by somatic cell genetic techniques.     

Duplication of an Xp segment that includes the ZFX locus causes sex inversion in man

Summary Two 46,XY females with tandem duplications of an X short arm segment were studied by cytogenetic and Southern blot analysis. The results show that the duplicated segment in each case included the Xp21.2–Xp22.2 interval, resulting in a double dose of ZFX on the single active X chromosome. The results from our two cases, in conjunction with those reported by other workers, lead us to conclude that the duplication is the reason for the sex inversion. If ZFY and ZFX are indeed sex-determining gene loci, these findings favour a model of sex determination characterized by antagonistic interaction between these genes.     

An antibody against secretogranin I (chromogranin B) is packaged into secretory granules

We have investigated the sorting and packaging of secretory proteins into secretory granules by an immunological approach. An mAb against secretogranin I (chromogranin B), a secretory protein costored with various peptide hormones and neuropeptides in secretory granules of many endocrine cells and neurons, was expressed by microinjection of its mRNA into the secretogranin I-producing cell line PC12. An mAb against the G protein of vesicular stomatitis virus--i.e., against an antigen not present in PC12 cells--was expressed as a control. The intracellular localization and the secretion of the antibodies was studied by double-labeling immunofluorescence using the conventional and the confocal microscope, as well as by pulse-chase experiments. The secretogranin I antibody, like the control antibody, was transported along the secretory pathway to the Golgi complex. However, in contrast to the control antibody, which was secreted via the constitutive pathway, the secretogranin I antibody formed an immunocomplex with secretogranin I, was packaged into secretory granules, and was released by regulated exocytosis. Our results show that a constitutive secretory protein, unaltered by genetic engineering, can be diverted to the regulated pathway of secretion by its protein-protein interaction with a regulated secretory protein. The data also provide the basis for immunologically studying the role of luminally exposed protein domains in the biogenesis and function of regulated secretory vesicles.     

Leaf thionins, a novel class of putative defence factors

Leaf thionins of barley have been identified as a novel class of highly abundant polypeptides with antifungal activity, which are present in walls and vacuoles of barley leaf cells. Similar thionins occur not only in monocotyledonous but also in various dicotyledonous plants. The leaf thionins of barley are encoded by a complex multigene family, which consists of at least 50–100 members per haploid genome. The toxicity of these thionins for plant pathogenic fungi and the fact that their synthesis can also be triggered by pathogens strongly suggest that leaf thionins are involved in the mechanism of plant defence against microbiol infection.     

Comparative measurements of the xylem pressure ofNicotiana plants by means of the pressure bomb and pressure probe

Determination of the pressure in the water-conducting vessels of intactNicotiana rustica L. plants showed that the pressure probe technique gave less-negative values than the Scholander-bomb method. Even though absolute values of the order of −0.1 MPa could be directly recorded in the xylem by means of the pressure probe, pressures between zero and atmospheric were also frequently found. The data obtained by the pressure probe for excised leaves showed that the Scholander bomb apparently did not read the actual tension in the xylem vessles ofNicotiana plants. The possibility that the pressure probe gave false readings was excluded by several experimental controls. In addition, cavitation and leaks either during the insertion of the microcapillary of the pressure probe, or else during the measurements were easily recognized when they occurred because of the sudden increase of the absolute xylem tension to that of water vapour or to atmospheric, respectively. Tension values of the same order could also be measured by means of the pressure probe in the xylem vessels of pieces of stem cut from leaves and roots under water and clamped at both ends. The magnitude of the absolute tension depended on the osmolarity of the bathing solution which was adjusted by addition of appropriate concentrations of polyethylene glycol. Partial and uniform pressurisation of plant tissues or organs, or of entire plants (by means of the Scholander bomb or of a hyperbaric chamber, respectively) and simultaneous recording of the xylem tension using the pressure probe showed that a 1∶1 response in xylem pressure only occurred under a few circumstances. A 1∶1 response required that the xylem vessels were in direct contact with an external water reservoir and/or that the tissue was (pre-)infiltrated with water. Corresponding pressure-probe measurements in isolated vascular bundles ofPlantago major L. orP. lanceolata L. plants attached to a Hepp-type osmometer indicated that the magnitude of the tension in the xylem vessels was determined by the external osmotic pressure of the reservoir. These and other experiments, as well as analysis of the data using classical thermodynamics, indicated that the turgor and the internal osmotic pressure of the accessory cells along the xylem vessels play an important role in the maintenance of a constant xylem tension. This conclusion is consistent with the cohesion theory. In agreement with the literature (P.E. Weatherley, 1976, Philos. Trans. R. Soc. London Ser. B23, 435–444; 1982, Encyclopedia of plant physiology, vol. 12B, 79-109), it was found that the tension in the xylem of intact plants under normal and elevated ambient pressure (as measured with the pressure probe) under quasi-stationary conditions was independent of the transpiration rate over a large range, indicating that the conductance of the flow path must be flow-dependent.     

A sensitive and rapid luminescence sandwich assay for antibodies to hepatitis-B surface antigen (Anti-HBsAg)

A chemiluminescent assay for hepatitis-B surface antigen is described which used an isoluminol derivative as the label. The assay is precise intra-assay CV, 1.96-2.45%; inter-assay CV, 5.26-8.11% and has a lower detection limit for hepatitis-B surface antigen of 1.3U/I.     

Characterization of the genetic system of the xylose-fermenting yeast Pichia stipitis

High mutant frequencies indicated that the wild-type strains of Pichia stipitis are haploid. Sporulation ability of these clones pointed to a homothallic life cycle. Mating was induced by cultivation under nutritionally poor conditions on malt extract medium. Conjugation was followed immediately by sporulation. However, hybrids could be rescued by transferring the nascent zygotes to complete medium before meiosis had started. Under rich nutritional conditions, hybrids were mitotically stable and did not sporulate. The segregation pattern of auxotrophic markers of diploid zygotes indicated regular meiosis, although asci contained preferentially spore dyads. Received: 29 February 1996 / Accepted: 29 March 1996     

Control of Photosystem II in spinach leaves by continuous light and by light pulses given in the dark

The light-induced induction of components of non-photochemical quenching of chlorophyll fluorescence which are distinguished by different rates of dark relaxation (qNf, rapidly relaxing and qNs, slowly relaxing or not relaxing at all in the presence brief saturating light pulses which interrupt darkness at low frequencies) was studied in leaves of spinach.After dark adaptation of the leaves, a fast relaxing component developed in low light only after a lag phase. Quenching increased towards a maximum with increasing photon flux density. This fast component of quenching was identified as energy-dependent quenching qE. It required formation of an appreciable transthylakoid pH and was insignificant when darkened spinach leaves received 1 s pulses of light every 30 s even though zeaxanthin was formed from violaxanthin under these conditions.Another quenching component termed qNs developed in low light without a lag phase. It was not dependent on a transthylakoid pH gradient, decayed exponentially with a long half time of relaxation and was about 20% of total quenching irrespective of light intensity. When darkened leaves were flashed at frequencies higher than 0.004 Hz with 1 s light pulses, this quenching also appeared. Its extent was very considerable, and it did not require formation of zeaxanthin. Relaxation was accelerated by far-red light, and this acceleration was abolished by NaF.We suggest that qNs is the result of a so-called state transition, in which LHC II moves after its phosphorylation from fluorescent PS II to nonfluorescent PS I. This state transition was capable of decreasing in darkened leaves the potential maximum quantum efficiency of electron flow through Photosystem II by about 20%.Abbreviations PFD photon flux density - PS photosystem     

Generation of rhythmic and arrhythmic behaviour of Crassulacean acid metabolism in Kalanchoë daigremontiana under continuous light by varying the irradiance or temperature: Measurements in vivo and model simulations

Leaves of Kalanchoë daigremontiana Hamet et Perr. at a photon flux density (PFD) above 220 mol·m^–2s^–1 (400–700 nm) or at leaf temperatures above 27.0 °C showed a rapid loss of rhythmicity, and a more or less pronounced damping-out of the endogenous circadian rhythm of CO₂ exchange under continuous illumination. This rhythm was reinitiated after reduction of the PFD by 90–120 mol·m^–2·s^–1 or reduction of leaf temperature by 3.5–11.0 °C under otherwise unchanged external conditions. The reduction in the magnitude of the external control parameter of the Crassulacean acid metabolism (CAM) rhythm (i.e. PFD or leaf temperature) set the phase of the new rhythm. The maxima of CO₂ uptake occurred about 5, 28, 51, 75 h after the reduction. Simulations with a CAM model under comparable conditions showed a similar behaviour. The influence of temperature on the endogenous CAM rhythm observed in K. daigremontiana in vivo could be simulated by incorporating into the model temperature-dependent switch modes for passive efflux of malate from the vacuole to the cytoplasm. Thus, the model indicates that tonoplast function plays an important role in regulation of the endogenous CAM rhythm in K. daigremontiana.Abbreviations CAM Crassulacean acid metabolism - PAR photosynthetically active radiation - PFD photon flux density This work was supported by a grant to F.B. and U.L. from Teilprojekt B5 in the Sonderforschungsbereich 199 of the Deutsche Forschungsgemeinschaft (Bonn, Germany) and by a grant to T. E. E. G. from the Sudienstiftung des deutschen Volkes (Bonn, Germany). Erika Ball is thanked for processing of time-course data for the analysis of Fourier spectra.