Paul L. Mann

Membrane Oligosaccharides: Structure and Function during Differentiation

Recent results gathered by normal light microscopy, immunocytochemistry, fluorescent-analog cytochemistry, and electron microscopy have allowed an improved interpretation of ameboid movement and related phenomena. 1. The contractile system responsible in Amoeba proteus for the generation of motive force for protoplasmic streaming and a large variety of dynamic activities is represented mainly by a thin cortical filament layer at the cytoplasmic face of the cell membrane (Fig. 18I). During normal locomotion this layer exhibits a distinct structural and physiological polarity with three different zones: a zone of reformation at the front (A), a zone of contraction in the intermediate cell region (B), and a zone of destruction at the uroid (C). 2. Two types of filaments participate in the formation of the cortical layer: (1) randomly distributed thin (actin) filaments exhibiting a parallel orientation in the anterior (Fc1) and a disordered arrangement in the intermediate and posterior cell region (Fc2; see also Fig. 17b), and (2) thick (myosin) filaments in close association with F-actin and mostly restricted to the intermediate and posterior cell region (Fc2). 3. The internal hydraulic pressure generated by localized active contraction of the cortical layer is transmitted to the endoplasm via the cell membrane and converted into directed streaming by a gel-sol gradient of decreasing viscosity between the uroid and the front. Calcium ions, ATP, and regulative proteins (profilin and a kinase) play an essential role in controlling both the interaction of actin and myosin and the sol-gel state of the cytoplasmic matrix. 4. Any alterations externally induced in the polarity of the cortical filament system by chemical or physical stimulation and inhibition cause immobilization of the amebas (Fig. 18II) with characteristic changes in (1) cell shape (spherulation and cell flattening), (2) membrane dynamics (cytotic and cytokinetic activities), and (3) cytoplasmic organization (hyalogranuloplasmic separation). pseudopodial tip (Fig. 18III, b----c, d----e), (3) destruction of the old layer at the hyalogranuloplasmic border (Fig. 18III, c,e), and (4) alternate solation (Fig. 18III, b and d) and gelation (Fig. 18III, c and e) of the hyaloplasm between the layer and the plasma membrane. The retraction of pseudopodia is accomplished by a local contraction of the cortical layer in conjunction with a simultaneous gel-sol transformation of the ectoplasmic cylinder. 6. The expression of a rather complex cytoskeleton which is composed not only of microfilaments and associated proteins, but also of intermediate- and microtubularlike structures has to be considered in future

Enhancement of abnormal tissue uptake of antibodies, tumor-specific agents or conjugates thereof for diagnostic imaging or therapy

Biomodulators, in conjunction with antibodies, tumor-specific agents or conjugates thereof, optionally linked to imaging-active moieties, can be administered to a host to enhance images thereof, e.g., NMR-, X-ray- or radioimages, preferably by increasing aberrant tissue signal intensity.

Cell surface oligosaccharide modulation during differentiation. I. Modulation of lectin binding.

IMR-90 human lung fibroblasts exhibit quantitative and qualitative changes in their cell surface oligosaccharide expression as a result of low-density growth, contact inhibition of growth and changes in population doubling level (PDL). Binding of Concanavalin-A (CON-A), wheat germ agglutinin (WGA) and Ricinus communis (RCA-120) was shown to decrease by 20-40% as the cells advanced through their lifespan (but significantly before morphological manifestations commonly associated with senescence, which occur at approximately PDL 45). The binding of Dolichos biflorus agglutinin (DBA) increased by 100% over the same time interval. These changes were detected by quantitation of FITC conjugated lectins on single cells and the use of a sensitive analytical biotin-avidin-enzyme amplification assay on whole populations of cells. These studies were performed on randomly growing cultures. These results suggest an important and dynamic role for the cell surface oligosaccharide in growth control and cellular senescence.

Cell surface oligosaccharide modulation during differentiation: III. Lectin affinity class distributions

In previous studies we showed that the onset of the morphological phenotype of cellular senescence (IMR-90) in vitro is preceded by complex cell surface changes. Using fluorescently conjugated lectins these studies showed: (1) quantitative PDL-dependent decreases in cell surface mannosyl, galactosyl, and N-acetyl-glucosamine residues; (2) these changes were correlated with changes in ligand/lectin membrane mobility, suggesting a functional relationship for the quantitative changes. To further investigate the biological significance of these observations we have developed a synthetic ligand competition assay to analyze the lectin binding event itself. The results of these analyses show that: (1) the number of binding affinity class distributions is highly restricted; (2) the PDL-dependent mannosyl change is due to the loss of a high-affinity class distribution without significant change in the low-affinity site; and (3) PDL-dependent changes in both galactosyl and N-acetyl glucosamine binding events are the result of changes in the affinity class distributions. These results are interpreted in terms of the potential available energy to act as the basis for signal transduction at the cell surface.

Cell surface oligosaccharide modulation during differentiation: IV. Normal and transformed cell growth control

In previous studies it was shown that cell surface oligosaccharide affinity class distributions and binding capacities were down-regulated as normal cells approach senescence. Using a sensitive, amplified, lectin/specific-ligand competition analysis three other growth regulation states were compared to that of cellular senescence. Non-senescent and senescent low-density and contact-induced growth inhibition was compared with neoplastic cell growth control. Non-senescent human fetal lung fibroblasts (IMR-90) down-regulated their mannosyl and galactosyl specificities in response to both low-density and contact-induced growth inhibition. Senescent IMR-90 down-regulate their mannosyl residues in response to contact conditions while they up-regulate their galactosyl residues under the same conditions. Growth-transformed transplantable canine glioma cells did not show density-dependent regulation of their cell surface oligosaccharide structures. Modulation of the CG cells with a specific alpha-mannosidase II inhibitor, Ricinus communis a galactosyl specific lectin, and pokeweed mitogen a cellular differentiating agent resulted in an altered growth phenotype and up-regulation of the mannosyl and galactosyl surface oligosaccharides. These data indicate a controller function for the cell surface oligosaccharides and a general influence on growth control.

Reduction of filamin in late passage human diploid fibroblasts (IMR-90)

Progressive subcultivation of IMR-90 cells results in non-proliferative, heterogeneous cultures which may reflect aging of the diploid line (Hayflick, Exp. Cell Res., 37 (1965) 614). We have observed that late passage cells exhibit different rates of spreading and morphogenesis when compared to early passage groups, phenomena which we attribute to altered reassembly of the cytoskeleton in senescent cells (Kelley et al. Mech. Ageing Dev., 13 (1980) 127). To determine whether potential differences in cytoskeletal proteins develop with progressive subcultivation, early and late passage cultures were extracted with 0.5% Triton X-100 for 1 min followed by 1.0% sodium dodecyl sulfate (SDS) prior to separation and characterization of extracted proteins by electrophoresis on 7.5-15% gradient SDS gels. Extractions were made of both culture groups 3, 6 and 24 h after reseeding. Cytoskeletal ultrastructure at each stage of spreading was examined either in replicas of extracted cells or directly by scanning electron microscopy. Although considerable variation in cytoskeletal organization was observed, qualitative differences in gel banding patterns of actin, myosin and tubulin were not apparent at selected time points. However, late passage cells at 6 h and 24 h did not exhibit filamin associated with the Triton insoluble fraction as did early passage cells. Since it has been demonstrated that filamin is capable of cross-linking actin microfilaments into bundles or sheets, we suggest that it is a principal element for the variant cell shape and cytoskeletal morphology observed during altered spreading behavior of late passage human diploid fibroblasts.

Image enhancement by coadministration of biomodulators and structurally modified imaging agents

Biomodulators can be administered together with an agent such as a drug or an imaging agent (specific or non-specific) structurally modified to take advantage of perturbations of cell oligosaccharide displays caused by biomodulators, to enhance images of a host, e.g., NMR-, X-ray- or radioimages, preferably by increasing aberrant tissue signal intensity. Biomodulators condition tissue to enhance or otherwise modify up-take of the drug or structurally modified imaging agent.

Cell surface oligosaccharide modulation during differentiation. II. Membrane mobility of oligosaccharide lectin conjugates

The quantitative, population doubling level (PDL) dependent changes in cell surface oligosaccharides on IMR-90 cells, were investigated from the perspective of membrane mobility of the lectin-oligosaccharide conjugates. Concanavalin-A (CON-A), wheat germ agglutinin (WGA), Ricinus communis agglutinin (RCA-120), and Dolichos biflorus agglutinin (DBA) were all observed to cluster, cap, and endocytose in cultured human diploid fibroblasts (IMR-90). Quantitative photometry at 37 degrees C over defined periods of time indicated that as the IMR-90s approached cellular senescence a specific lectin-dependent inability to either endocytose or process the capped complex occurred. The development of a biotin/avidin/enzyme amplification assay permitted the assignment of the accumulating signal to the internal compartment. Kinetic data indicate that there are at least three separate (and separable) mechanisms for the PDL related changes in lectin binding. Data for the CON-A complex indicates that at least two classes of functional complexes are present. Regression analysis of the kinetic data for the RCA-120 complex indicates a similar membrane clearance for the IMR-90s at all population doubling levels (PDL), suggesting that the quantitative differences observed earlier were due to simple quantitative reductions in the RCA complexing molecules. Data for WGA mobility on the membrane indicates that they are both changes in the number and mobility status of the complexes. These results indicate that the quantitative changes in lectin binding observed previously as IMR-90 cells approach senescence are correlated with alterations in membrane mobility patterns of the lectin oligosaccharide conjugates.

Biomodulation: An integrated approach to access and manipulate biological information

The concept of altering host/tumor responsiveness (surveillance) as a specific tool in the management of neoplastic disease has been the subject of both basic science and clinical studies for many years. One of the first practical applications of this approach was described by Von Leyden and Blumenthal in 1902. In this study autologous tumor cells were injected into a patient as part of an intervention protocol. In 1908 Paul Ehrlich predicted the existence of a host immuno-dependent protective mechanism. Burnet formalized this concept in 1970. The first element of Burnet’s proposal is the existence of unstable cellular clones with neoplastic potential. These clones are a normal part of mammalian existence and are important in the maintenance of surveillance mechanisms. The second element is the proposal that the host immune system is designed to hold these clones in stasis. These two inter-related elements laid the conceptual groundwork for the theory that the “induction” of the immune system could be beneficial in cases where the normal stasis mechanisms have failed and a neoplasm has expressed. Thus, from its inception these two elements were given equal weight. The first is termed by us immuno-regulatory, with the implication of recognition and regulation [both proliferative and differentiative] components. The second is termed immuno-defensive, with its implication of effector functions involved in lytic actions. In recent years these separate concepts have been apparently collapsed into the search for enhanced specificity in immune-defensive functions. Thus the modern day applications of Burnet’s concept have evolved and the field of Biological Response Modifiers [BRMs] was created.

Aldose reductase expression and prostaglandin E2 production are coordinately regulated in cultured rat mesangial cells

There is increasing evidence that a link between the polyol pathway and prostaglandins is important in the pathogenesis of diabetic nephropathy. The presence of the polyol pathway in the kidneys of normal animals, the galactose-fed rat, and animals with experimental diabetes has been established. While aldose reductase (AR) immunoreactive protein (AR-IRP) and AR mRNA are expressed at high levels in renal medulla, the sites of AR synthesis and regulation and metabolic consequences of AR activity in renal cortex are uncertain. The present study was conducted to test the hypothesis that AR expression and PGE2 production are coordinately regulated in glomerular mesangial cells. To test this hypothesis, we measured AR-IRP, AR mRNA, and PGE2 production in mesangial cells isolated from rats maintained on diets containing normal chow (MC-N), 50% galactose (MC-G), and 50% dextrin (MC-D). The rank order for each parameter studied (AR-IRP, AR mRNA, PGE2) was MC-N > MC-G > MC-D. Western blot analysis demonstrated that MC-N (optical density [OD] 1.0), MC-G (OD 0.59), and MC-D (OD 0.25) express AR-IRP. Slot-blot analyses demonstrated that levels of AR mRNA were greatest in MC-N (1.0), intermediate in MC-G (0.49), and lowest in MC-D (0.31). Ribonuclease (RNase) protection analyses demonstrated a similar pattern of AR mRNA expression, with MC-N at 1.0, MC-G at 0.60, and MC-D at 0.33. PGE2 production (pg/5 x 10(4) cells/30 min) was highest in MC-N (278 +/- 29), intermediate in MC-G (110 +/- 9), and lowest in MC-D (37 +/- 4).(ABSTRACT TRUNCATED AT 250 WORDS)