Deborah L. Berglund

Isolation of viable tumor cells following introduction of labelled antibody to an intracellular oncogene product using electroporation

A method for labelling the intracellular ras oncogene product, p21, with a monoclonal antibody, in B16BL6 mouse melanoma cells for subsequent flow cytometric analysis and viable cell sorting is described. Permeabilization of the cells for introduction of labelled antibody was attempted using (1) lysolecithin treatment, and (2) electroporation, a much more highly controllable technique. Permeabilization was assessed using propidium iodide or calcofluor white M2R staining, while short-term cellular viability was determined using fluorescein diacetate staining and long-term viability by reculturing the sorted cells. We successfully introduced labelled antibody into the cells with both permeabilization techniques. Insufficient numbers of viable permeabilized cells were obtained lysolecithin treatment to warrant an attempt at viable cell sorting. On the other hand, good numbers of viable, permeabilized cells were obtained using electroporation and we successfully sorted viable tumor cell populations based on the intensity of their anti-p21ras staining. These sorted tumor cells retained their characteristic anti-p21ras staining intensity for at least 2 weeks of propagation in culture.

CELL SURFACE AND SUBSTRATE DISTRIBUTION OF THE 67-KDA LAMININ-BINDING PROTEIN DETERMINED BY USING A LIGAND PHOTOAFFINITY PROBE

BACKGROUND Peptide 11, a nine-amino acid sequence from the beta1 chain of laminin-1, has been reported to inhibit tumor cell invasion of basement membranes, and to reduce tumor lung colonization (Iwamoto et al.: Science 238:1132-1134, 1987; Landowski et al.: Clin Exp Metastasis 13:357-372, 1995). The peptide is a ligand for the 32/67-kDa laminin-binding protein (LBP); however, the mechanism by which the 67-kDa LBP promotes invasion is unknown. METHODS We have synthesized a highly specific probe for the 67-kDa LBP by adding a biotinylated residue, and replacing the required tyrosine in peptide 11 with the photoactivatable bezophenone crosslinker, 4-benzoyl-L-phenylalanine. This probe was used to follow the distribution of the 67-kDa LBP by gel electrophoresis, fluorescence-activated cell scanning, and confocal microscopy techniques. RESULTS A single crosslinked protein, consistent with the high molecular weight form of the LBP, was found on Western blots of membrane detergent extracts from cells treated with the ligand probe. A CHO cell line, manipulated to overexpress the laminin-specific alpha6beta1 integrin, exhibited increased invasiveness, and expressed more cell surface 67-kDa LBP. Membrane-associated 67-kDa LBP was found in the vicinity of focal adhesion plaques and also associated with the matrix substrate. Studies on conditioned medium indicated that the matrix-associated LBP derived from material that was shed from the cells, with more being shed from the more invasive CHO variants. CONCLUSIONS These results demonstrate the utility of this novel probe in diverse experimental protocols, and suggest that shedding of the 67-kDa LBP may have a role in promoting tumor cell invasion.

Cell–cell and cell–surface interactions in an illuminated biofilm: Implications for marine sediment stabilization†

Most wetted surfaces that are illuminated support a population of phototrophs. The marine sediment is no exception and there the major component of the microphytobenthic population is diatoms. These organisms are credited with stabilizing the sediment against physical disturbance by virtue of the extracellular carbohydrate polymers that they elaborate. However, diatoms synthesize and secrete several carbohydrate polymers and it is not certain which of them is involved in the stabilization process. In order to investigate this, we have constructed small glass bead-filled flow through bioreactors to mimic marine sediments. The flow rate through the bioreactors was found to reflect the physical stability of the bead bed. Thus flow rate was measured as a function of diatom growth and the production of three operationally-defined polymers, i.e., those soluble in the medium, those soluble in 0.5 M NaHCO3 at 90 °C and those not soluble in either solvent (matrix polymer). Growth of the diatoms did not change the hydraulic conductivity of the bioreactors. For Amphora coffeaeformis, neither did the production of medium-soluble nor NaHCO3-soluble polymers. However, matrix polymer accumulation was directly correlated with a reduction in flow (regression coefficient R2 = 0.96) and stabilization against physical disturbance. Results with species of Navicula were not as clear. Both NaHCO3-soluble and matrix polymers were involved in producing the flow reduction. In the same manner we also measured the effect of Pseudoalteromonas haloplanktis growth on bead bed hydraulic conductivity and bead bed stability. Growing alone, no effect was found, but in co-culture with a single diatom species, the bacteria reduced the diatom effect on flow through the bioreactors seen earlier, however did not reduce the extent of their growth. Confocal scanning laser microscopy of beads colonized with diatoms alone, or diatoms in co-culture with bacteria, revealed that P. haloplanktis was able to inhibit diatom adhesion to the beads. When the bacteria were present there was less matrix polymer evident. We speculate that this interference with diatom metabolic activity was either the result of less matrix polymer synthesis, or its hydrolysis by the bacteria. The results are applicable to mixed species biofilms of this type on surfaces other than sediments.

Flow cytometry as a method for assaying the biological activity of phytotoxins

Abstract A flow cytometric method has been developed for the qualitative and quantitative evaluation of the biological activities of phytotoxins from plant pathogenic fungi. The method utilized fresh wheat ( Triticum aestivum L.) leaf protoplast preparations treated with purified phytotoxins, triticone A-B and triticone D. Subsequently, protoplasts were exposed to fluorescein diacetate, and analyzed by flow cytometry. Information acquired included fluorescence owing to esterase activity on fluorescein diacetate, and chlorophyll autofluorescence. Results indicate that triticone A-B has a rapid dose-dependent toxic effect on wheat protoplasts but triticone D has no toxic effect. This method can also yield information on the mechanism of action of phytotoxins that are relatively unstable or available only in small quantities.

Triticone A: a novel bioactive lactam with potential as a molecular probe.

Triticone A is one member of a family of novel compounds which are spirocyclic lactams produced by several plant pathogenic fungi including Drechslera tritici repentis on wheat. It undergoes racemization to form triticone B and when tested, the enantiomeric mixture causes chlorosis and necrosis on a wide range of plants. Fluorescein diacetate treated protoplasts in conjunction with various triticone treatments allowed for accurate quantitation of the biological activity of the toxin. Various physiological functions of the wheat cell are impaired including the Hill and CO2 fixation reactions in photosynthesis. In addition, triticone A inhibits enzymes that have SH functional groups as part of their active site, eg., the protease-ficin. Neither triticone C or D had any activity in the enzyme or protoplast assays. It is apparent that triticone A has some potential as a molecular probe in a variety of biological systems.

Isolation of protoplasts from loblolly pine needles and their flow-cytometric analysis for air pollution effects

Abstract Flow-cytometric analysis was used for the first time to determine the effects of air pollutants on plant biochemistry. The methods used were exploratory, but the results indicate that the technique is a valuable tool for researchers interested in the effects of air pollutants on vegetation. Evidence of carryover effects was seen in protoplasts isolated from new needles of 2-year-old loblolly pines ( Pinus taeda L.) which had been treated with ozone and simulated acidic rain the preceding year. Overall, staining of the protoplasts with constituent-specific fluorescent dyes showed decreased levels of fluorescence for esterase activity, protein, neutral lipids, and RNA in the ozone-treated samples compared with the charcoal-filtered or ambient samples. However, within pollutant treatments, low-level shifts in biochemistry were seen; for instance, simulated acidic rain at pH 5.0 ameliorated the effects of ozone on neutral lipids and RNA, and at pH 4.3 ameliorated the effects of ozone on protein. This research provides evidence at the biochemical level that pollutant treatments given in one year may be reflected in the succeeding years growth. Image analysis of cross sections of fresh young pine needles supported the biochemical data.

A flow cytometric method for intracellular labeling and purification of rare neuronal populations: isolation of fixed neurophysin neurons.

A method is described for flow cytometric analysis and fluorescence-activated cell sorting of small populations of neurons following dissociation of fixed brain tissue and immunofluorescent labeling of intracellular antigens. This method has been successfully applied to neurophysin-containing magnocellular neurons of the rat supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. These neurons constitute a rare population in the context of flow cytometry, comprising less than 2% of all cells present in dissociated tissue punches of SON and PVN. Following labeling with anti-neurophysins sera and fluorescein-conjugated second antibody, a highly enriched population containing 80-85% neurophysin-positive neurons was isolated by fluorescence-activated cell sorting. Recovery of 29% of all neurophysin-containing neurons in the SON/PVN was achieved. Perikarya were recovered largely intact, frequently with attached proximal dendritic processes. Applications of this method include purification of specific neuronal types for use as immunogens in production of monoclonal antibodies to cell-type-specific antigens, and rapid surveys of fluorescent lectin or other ligand binding to cell populations identified by the presence of particular intracellular antigens.