Cells In Intact Tissue Research Articles

One Protoplast Is Not the Other!

Protoplasts are widely used to study a variety of cellular processes because they are easy to transform, manipulate and image at high resolution. Protoplasts are thought to loose their original cell identity and therefore their origin is of minor unimportance. Hence, leaf mesophyll protoplasts are generally used to study processes active in other cell-types. However, there is no consensus whether data from protoplasts hold true for cells in intact tissues. We developed a procedure to isolate protoplasts from petals and compared their features with those of leaf protoplasts and intact tissues. Petal protoplasts derived from epidermis or mesophyll can be easily distinguished by their color and accurately reproduce the cell-specific differences in gene regulation and protein sorting observed in the intact tissue. We show that protoplasts maintain the features of the original cells in the intact tissue and, depending on their origin may produce completely different results in gene expression or protein localization assays. Consequently, analyses in leaf mesophyll protoplasts of genes or proteins that are expressed in other cell-types should be interpreted with caution. We conclude that with a few simple adjustments, to distinguish different cell-types, protoplasts can provide an excellent tool to reliably analyze highly cell-specific processes.

1607 HIGH-RESOLUTION DETECTION OF PTEN GENOMIC DELETION BY FLUORESCENCE IN SITU HYBRIDIZATION (FISH) USING 330 FIXED PARAFFIN-EMBEDDED TISSUE SAMPLES

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 20111607 HIGH-RESOLUTION DETECTION OF PTEN GENOMIC DELETION BY FLUORESCENCE IN SITU HYBRIDIZATION (FISH) USING 330 FIXED PARAFFIN-EMBEDDED TISSUE SAMPLES Jeremy Squire, Olga Ludkovski, Dave DeGrace, Andrew Evans, Kanishka Sircar, Tarek Bismar, and Maisa Yoshimoto Jeremy SquireJeremy Squire Kingston, Canada More articles by this author , Olga LudkovskiOlga Ludkovski Toronto, Canada More articles by this author , Dave DeGraceDave DeGrace Kingston, Canada More articles by this author , Andrew EvansAndrew Evans Toronto, Canada More articles by this author , Kanishka SircarKanishka Sircar Houston, TX More articles by this author , Tarek BismarTarek Bismar Calgary, Canada More articles by this author , and Maisa YoshimotoMaisa Yoshimoto Kingston, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1715AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Deletion of a tumour suppressor gene in routine formalin-fixed paraffin-embedded (FFPE) pathology sections is readily assayed by fluorescence in situ hybridization (FISH). However, the sectioning of the FFPE tissue samples often results in truncation artefacts of interphase nuclei leading to partitioning of the spherical shaped nucleus. As many as 20–40% of nuclei may be have been partially severed through the sectioning process leading to “false positive” signal losses and considerable interpretative difficulties. In this study a novel probe combination, for detecting gene deletions with high specificity was developed. METHODS Four color FISH probes were designed to detect genomic losses in the PTEN genomic region in 5 micron FFPE tissue sections. We evaluated the reliability of this assay using FFPE CaP tissue samples (tissue microarrays, n=330 patients), and mapping the breakpoint regions associated with PTEN deletions in this cohort derived from two institutions. RESULTS The probe combination selected reduced the rate of signal losses due to nuclear truncation by 50%. 132/330 (40%) of patient tumours were deleted for PTEN. The extent of deletion in each of the 132 tumors was mapped by FISH and the major class (41%) had an interstitial genomic deletion interval of 2Mb or less. The second most frequent class (28% of deleted tumors) involved genomic losses that were more heterogeneous in length, and involved PTEN deletions accompanied by adjacent genes on either side of the locus, with more losses extending in a telomeric direction. CONCLUSIONS FISH-based tumor suppressor deletion assay offers the advantage of highly specific and statistically significant quantitative evaluation of the gene locus of interest within individual cells in intact tissue. Consequently, CaP samples can be thoroughly characterized in an attempt to identify any PTEN heterogeneity, within or between foci in multifocal tumors. The clinical value of this assay is that presence of PTEN deletion by FISH is associated with earlier biochemical recurrence and increased mortality. For tumors with a homozygous deletion there is a strong association with hormone refractory CaP and metastatic disease. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e645 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jeremy Squire Kingston, Canada More articles by this author Olga Ludkovski Toronto, Canada More articles by this author Dave DeGrace Kingston, Canada More articles by this author Andrew Evans Toronto, Canada More articles by this author Kanishka Sircar Houston, TX More articles by this author Tarek Bismar Calgary, Canada More articles by this author Maisa Yoshimoto Kingston, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Ryanodine Receptors Outside of Couplons are Involved in Excitation-Contraction Coupling in Rabbit Ventricular Myocytes

Current theories of excitation-contraction coupling (ECC) in ventricular myocytes assert that L-type Ca channels interact with clusters of ryanodine-sensitive Ca release channels (RyRs) within couplons. We hypothesized that RyR clusters exist also outside of couplons and contribute to ECC. We investigated this hypothesis by three-dimensional imaging of RyR clusters and sarcolemma of isolated myocytes lying flat (horizontal) and on end (vertical). We deconvolved the image stacks, created reconstructions of cell segments, and identified RyR cluster types. RyR clusters remote to sarcolemma were assumed to be outside of couplons. Similar studies were performed on intact ventricular tissue. Furthermore, we imaged evoked Ca transients and sarcolemma of horizontal cells labeled with fluo-4 and di-8-anepps. Image sequences were acquired using rapid two-dimensional scanning (Zeiss LSM5Live, rates up to 300HZ). The image sequences were corrected for bleaching and cross-talk. In horizontal and vertical isolated cells, RyR clusters appeared to be arranged in sheets in the vicinity of Z-disks. Some RyR clusters were associated with sarcolemma, in particular transverse tubules, and are presumably part of couplons. However, most RyR clusters were not. Examination of cells in intact tissue revealed a smaller number of RyR clusters not associated with sarcolemma than in isolated cells. The density of transverse tubules was higher than in isolated cells. This loss of transverse tubules might be caused by the isolation procedure. Analysis of the rapid image sequences indicated that both types of RyR clusters were activated during an action potential. However, the RyR clusters not associated with sarcolemma were activated with delays of up to 10ms. In conclusion, we demonstrated that RyR clusters outside of couplons are involved in ECC. We suggest that activation of RyR clusters outside of couplons occurs by a common pool mechanism.

Hypoxia Differently Modulates Gene Expression of Inositol 1,4,5‐Trisphosphate Receptors in Mouse Kidney and HEK 293 Cell Line

Hypoxia is a state of insufficient oxygen supply of the tissue or cell. Kidney tissue is highly sensitive to oxygen deprivation and easily develops renal ischemic injury. Calcium transporters very sensitively react to oxygen deficiency. We investigated whether hypoxia affects the gene expression of intracellular calcium transporters in the intact kidney, and we compared the response to that of HEK 293 cells. Our results showed that, while in mouse kidney tissue hypoxia elevates mRNA for inositol 1,4,5-trisphosphate receptors (IP3R) type 1 (IP3R1) and ryanodine receptors (RyR) type 2 (RyR2), in culture of HEK 293 cells the gene expression of all IP3Rs decreased without affecting viability of the cells. RyR2 mRNA in HEK 293 cells was not significantly changed, but RyR1 gene expression was significantly increased by hypoxia. The different response of kidney tissue and HEK 293 cells to hypoxia could be due to unequal differentiation state of the cells in intact tissue and cultured embryonic cell line. The physiological relevance of this observation remains to be determined.

Laser scanning cytometry in human brain slices

The Laser Scanning Cytometry (LSC) offers quantitative fluorescence analysis of cell suspensions and tissue sections. We adapted this technique to immunohistochemical labelled human brain slices. We were able to identify neurons according to their labelling and to display morphological structures such as the lamination of the entorhinal cortex. Further, we were able to distinguish between neurons with and without cyclin B1 expression and we could assign the expression of cyclin B1 to the cell islands of layer II and the pyramidal neurons of layer V of the entorhinal cortex in Alzheimer's disease effected brain. In addition, we developed a method depicting the three-dimensional distribution of the cells in intact tissue sections. In this pilot experiments we could demonstrate the power of the LSC for the analysis of human brain sections.

Optical imaging of cervical pre-cancers with structured illumination: An integrated approach

Objective. Structured illumination microscopy is an inexpensive alternative to confocal microscopy that allows optical sectioning at a sub-cellular resolution. However, its application in imaging biological tissue has been limited by inadequate contrast present in them especially in reflectance imaging. Novel, optically active contrast agents like gold nanoparticles and quantum dots targeted against biomarkers of cancer can be integrated with structured illumination to image both the morphological and biochemical changes associated with epithelial pre-cancers. Methods. We modified the optical path of a widefield microscope to implement structured illumination both in reflectance and fluorescence modes. For imaging, we used 25-nm-diameter gold nanoparticles and CdSe quantum dots for reflectance and fluorescence imaging, respectively, to label three-dimensional tissue constructs of SiHa cervical cancer cells. Contrast agents were targeted against the epidermal growth factor receptor (EGFR) using an anti-EGFR monoclonal antibody. Agents targeted with a non-specific IgG antibody served as a control to monitor non-specific labeling. Results. Our result shows that optically sectioned images taken with structured illumination are very comparable to those obtained using confocal microscopy. Moreover, images of three-dimensional cultures stained with the anti-EGFR agents show significantly more image intensity than those stained with the IgG targeted control. Conclusion. Our findings suggest that the combination of novel optical contrast agents and structured illumination can differentiate neoplastic cells which overexpress EGFR from normal cells in intact tissue. Combining structured illumination microscopy with novel contrast agents can potentially provide a powerful and inexpensive tool to aid in the detection of cervical pre-cancers.

PAX6 expression identifies progenitor cells for corneal keratocytes.

Keratocytes of the corneal stroma produce a transparent extracellular matrix required for vision. During wound-healing and in vitro, keratocytes proliferate, becoming fibroblastic, and lose biosynthesis of unique corneal matrix components. This study sought identification of cells in the corneal stroma capable of assuming a keratocyte phenotype after extensive proliferation. About 3% of freshly isolated bovine stromal cells exhibited clonal growth. In low-mitogen media, selected clonal cultures displayed dendritic morphology and expressed high levels of keratan sulfate, aldehyde dehydrogenase 3A1, and keratocan, molecular markers of keratocyte phenotype. In protein-free media, both primary keratocytes and selected clonal cells aggregated to form attachment-independent spheroids expressing elevated levels of those marker molecules. The selected clonal cells exhibited normal karyotype and underwent replicative senescence after 65-70 population doublings; however, they continued expression of keratocyte phenotypic markers throughout their replicative life span. The progenitor cells expressed elevated mRNA for several genes characteristic of stem cells and also for genes expressed during ocular development PAX6, Six2, and Six3. PAX6 protein was detected in the cultured progenitor cells and a small number of stromal cells in intact tissue but was absent in cultured keratocytes and fibroblasts. Cytometry demonstrated PAX6 protein in 4% of freshly isolated stromal cells. These results demonstrate the presence of a previously unrecognized population of PAX6-positive cells in adult corneal stroma that maintain the potential to assume a keratocyte phenotype even after extensive replication. The presence of such progenitor cells has implications for corneal biology and for cell-based therapies targeting corneal scarring.

Calcium imaging of gut activity.

The major cell types regulating gut motility include enteric neurones, interstitial cells of Cajal (ICC) and their effector smooth muscle cells. These cells are arranged conveniently in nested layers through the gut wall. Our knowledge of how many of these cells in each layer are integrated to produce the various patterns of motility is largely unknown. So far, much of our knowledge of gut motility has usually been obtained by examining point sources of activity (e.g. intracellular recordings from enteric neurones, ICC and smooth muscle cells), rather than the spread of activity through these spatially distributed nerve and ICC networks, or smooth muscle syncitia. Our understanding of how these cells are integrated to produce gut movements would be greatly enhanced if we could image the activity in many of these cells in each layer, or many cells in several layers, simultaneously. Calcium (Ca2+) is a major signalling and regulatory molecule in most cells. In fact, electrical excitability in enteric neurones, ICC and smooth muscle is associated with robust rises in intracellular Ca2+ that long outlast the electrical events (e.g. action potentials in neurones and smooth muscle) that gave rise to them. These prolonged Ca2+ responses, together with the development of several high quality Ca2+ indicators, has provided a unique opportunity to image many cells in intact tissues simultaneously using ICCD video-rate cameras along with conventional microscopy. However, confocal microscopy has also been used, and has several advantages over the above systems. These include reduced photo-toxicity and bleaching and the elimination of out of focus light from different layers within the tissue. So far, despite some limitations with the calcium imaging techniques, the spread of activity through the two layers of smooth muscle, ICC networks and myenteric neurones in intact preparations, or cultured myenteric neuronal networks, is beginning to yield exciting new data about how these different cells interact and process information.

Photochemical gating of heterologous ion channels: remote control over genetically designated populations of neurons.

Heterologous proteins capable of transducing physical or chemical stimuli into electrical signals can be used to control the function of excitable cells in intact tissues or organisms. Restricted genetically to circumscribed populations of cellular targets, these selectively addressable sources of depolarizing current can supply distributed inputs to neural circuits, stimulate secretion, or regulate force and motility. In an initial demonstration of this principle, we have used elements of a G protein coupled signaling system, the phototransduction cascade of the fruit fly, to sensitize generalist vertebrate neurons to light [Zemelman, B. V., Lee, G. A., Ng, M. & Miesenböck, G. (2002) Neuron 33, 15-22]. We now describe the use of ectopically expressed ligand-gated ion channels as transducers of optical or pharmacological stimuli. When either the capsaicin receptor, TRPV1, the menthol receptor, TRPM8, or the ionotropic purinergic receptor P2X(2) was introduced into hippocampal neurons, the cells responded to pulsed applications of agonist with characteristic sequences of depolarization, spiking, and repolarization. Responses required cognate matches between receptor and agonist, peaked at firing frequencies of approximately 40 Hz, initiated and terminated rapidly, and did not attenuate. Precise dose-response relationships allowed current amplitudes and firing frequencies to be tuned by varying the concentration of ligand. Agonist could be administered either pharmacologically or, in the cases of TRPV1 and P2X(2), optically, through photorelease of the active compounds from the respective "caged" precursors, 4,5-dimethoxy-2-nitrobenzyl-capsaicin and P(3)-[1-(4,5-dimethoxy-2-nitrophenyl)ethyl]-ATP.

Oligodendrocyte precursor cells: reactive cells that inhibit axon growth and regeneration.

Oligodendrocyte precursor cells (OPCs) are a newly recognized glial component of the adult central nervous system of unknown function. Antibodies against the NG2 chondroitin sulfate proteoglycan have been useful tools to identify these cells in intact tissue. Here we review studies that show that OPCs react to several types of experimentally induced brain injury. Injury stimulates OPCs to re-enter the cell cycle, divide, and accumulate at the site of damage. OPCs, together with microglia and astrocytes, form the glial scar. Glial scars are thought to inhibit or prevent axonal regeneration and reactive OPCs contribute to this inhibition by producing growth-inhibiting chondroitin sulfate proteoglycans, particularly NG2. In developing animals, NG2 is found in areas, such as the perinotochordal mesenchyme, that are avoided by growing motor and sensory axons. Within the developing CNS, NG2-expressing cells surround the developing optic chiasm and tract and separate it from the overlying diencephalon. Thus, NG2-expressing cells are well positioned to inhibit axonal growth from developing as well as regenerating neurons.

Single-Cell Electroporationfor Gene Transfer In Vivo

We report an electroporation technique for targeting gene transfer to individual cells in intact tissue. Electrical stimulation through a micropipette filled with DNA or other macromolecules electroporates a single cell at the tip of the micropipette. Electroporation of a plasmid encoding enhanced green fluorescent protein (GFP) into the brain of intact Xenopus tadpoles or rat hippocampal slices resulted in GFP expression in single neurons and glia. In vivo imaging showed morphologies, dendritic arbor dynamics, and growth rates characteristic of healthy cells. Coelectroporation of two plasmids resulted in expression of both proteins, while electroporation of fluorescent dextrans allowed direct visualization of transfer of molecules into cells. This technique will allow unprecedented spatial and temporal control over gene delivery and protein expression.

Immunolabeled microtubules and microfilaments are visible in multiple cell layers of rye root tip sections

A protocol was developed to observe plant microtubules and actin microfilaments in large tissue samples without physical sectioning. Rye (Secale cereale L. cv. Rymin) root tip pieces from two-day-old seedlings were fixed and processed for immunolabeling. Incubation times of 24–48 h were required to insure adequate penetration of fixatives, antibodies, and washing buffers. Clearing of the tissue with methyl salicylate reduced background auto-fluorescence that would otherwise interfere with the resolution of cytoskeletal structures. Microtubules or microfilaments in 5–7 cell layers were visualized using the optical-sectioning capability of laser scanning confocal microscopy (LSCM) and projected as three-dimensional images. The three-dimensional character of the cytoskeletal elements is retained when viewing stained cells of intact tissue. The net-like character of a microfilament array radiating out from a single point into the cytoplasm is maintained when the cells are stained in intact root tip pieces and imaging is accomplished in situ.

Ca2+ waves are organized among hepatocytes in the intact organ

Hormone-induced increases in cytosolic Ca2+ (Cai2+) begin as Cai2+ waves in cells isolated from most types of tissue (1, 11), but whether such waves actually occur in vivo is unknown. To investigate this, we examined vasopressin-induced Cai2+ signals in hepatocytes within the perfused rat liver. Using confocal fluorescence video microscopy, we found that increases in Cai2+ began as waves that usually originated in hepatocytes near central venules, then spread opposite to the direction of blood flow, to hepatocytes near portal venules. We used immunochemistry to determine that the liver vasopressin V1a receptor is most concentrated among hepatocytes in the pericentral region, providing the mechanism by which Cai2+ waves originate there. Pericentral-to-periportal Cai2+ waves may direct peristaltic flow of bile, since Cai2+ induces contraction of the apical pole of hepatocytes and since peristaltic contractions in liver also occur in a pericentral-to-periportal direction. The organization of Cai2+ waves among cells in intact tissue may be a means by which an integrative, organ-level response is provided in response to hormonal stimuli.

Immunofluorescence localisation of microtubules in plant root tips embedded in butyl-methyl methacrylate

Most immunofluorescence studies of microtubules in plant cells have used enzymatically isolated cells whose position in the organ and stage of development was unknown. Moreover, attempts to label microtubules in cells in intact tissue have suffered from poor resolution and inferior tissue preservation. To overcome these difficulties, I have used a removable embedding resin to localise microtubules in situ. Tissues were fixed in 4% paraformaldehyde and 0.2% glutaraldehyde and embedded in butyl-methyl methacrylate. Semi-thin sections were extracted with acetone to remove the resin and labelled with anti-tubulin followed FITC-labelled second antibody. Brightly stained microtubule arrays were clearly visualized. This reversible embedding medium should provide a powerful tool to study developmental changes in microtubule arrays in growing and differentiating plant tissues.

Localization of somatomedin-C binding to bovine growth-plate chondrocytes in situ.

The somatomedins are a family of low-molecular-weight peptides that are thought to mediate the stimulatory effect of growth hormone on skeletal growth. The cells that are directly responsible for skeletal growth are the chondrocytes of the epiphyseal growth plate, and these are the presumed skeletal target cells for somatomedin. As with other peptide growth factors, the cellular effects of the somatomedins are initiated by the interaction of the growth factor with specific receptors on target-cell surface membranes. Chondrocytes that have been isolated from bovine growth plates were previously found to possess specific surface receptors for the principal growth-hormone-dependent somatomedin, somatomedin-C. These studies indicated that the interaction of growth-plate chondrocytes with somatomedin-C involves specific receptor-binding followed by somatomedin-C internalization by the cell, a process identical to that identified in the mechanism of action of other peptide growth factors in other cells. These studies, however, left unanswered the questions of whether there are differences in binding of somatomedin-C by the different cell populations within the physis and whether somatomedin-C has access to cells in intact tissues. The current studies address these issues and indicate that bovine physeal chondrocytes in situ are accessible to exogenous somatomedin-C, that they specifically bind somatomedin-C in situ, and that cells of different physeal zones bind somatomedin-C differently. Labeled somatomedin-C is specifically bound by cells of all physeal zones. However, the binding is greatest for those cells undergoing active synthesis of DNA in the proliferative zone.(ABSTRACT TRUNCATED AT 250 WORDS)

The number of synaptic boutons terminating on Xenopus cardiac ganglion cells is directly correlated with cell size.

The relationship between the size of parasympathetic neurones and the number of synaptic boutons terminating upon them has been studied in the cardiac ganglion of Xenopus laevis. Synaptic boutons were visualized by impregnation with zinc iodide and osmium (ZIO), which by electron microscopy was shown to stain heavily all synaptic boutons in six preparations. Light microscopic examination of the unipolar ganglion cells in intact tissue reveals that larger neurones have more synaptic boutons. The number of boutons terminating on the cell body is significantly correlated with its surface area. By statistical means it was possible to demonstrate that the relation between bouton number and surface area is linear and that the regression line has a y-intercept not significantly different from zero. Therefore the density of synaptic boutons, one per 127 micron2 of cell body surface, is independent of cell size. The size of synaptic boutons, measured as the area of apposition between bouton and cell body, is similar for small and for large ganglion cells; thus a constant fraction (2%) of the cell body on average is covered by synaptic boutons, regardless of cell size. The correlation between bouton number and cell body surface area is not the result of interaction between boutons. The frequency distribution of boutons per normalized cell was found to be similar to that expected from the Poisson distribution. Thus the probability that a bouton will be 'assigned' to a particular cell is independent of how many other boutons are present. The only factor that appears to influence the number of boutons on the cell body is its size.

Schwann cells of the olfactory nerves contain glial fibrillary acidic protein and resemble astrocytes

—Two antisera to glial fibrillary acidic protein from human brain and an antiserum to a 49 k dalton glial filament protein from human brain detected a cross-reacting antigen in the Schwann cells of the olfactory and vomeronasal nerves. The antigen was demonstrated at light- and electron-microscope levels. It was found throughout the cytoplasm and in association with cytoplasmic filaments of olfactory nerve Schwann cells in intact tissue and in Schwann cells grown in vitro. This observation, together with observations on the ultrastructure of olfactory nerve Schwann cells, relates them to central astroglia and to glial cells of the myenteric plexus, rather than to Schwann cells of other peripheral nerves. The unusual properties of olfactory nerve Schwann cells are of interest in relation to the regenerative abilities of the olfactory nerves.

Role of secretory component, a secreted glycoprotein, in the specific uptake of IgA dimer by epithelial cells.

The binding of secretory component (SC) to epithelial cells and its role in the specific uptake of immunoglobulin A (IgA) dimer has been studied in rabbit mammary gland and liver. SC, Mr approximately 80,000, secreted by epithelial cells of the mammary gland was found associated with the cell surface of mammary cells in intact tissue. Dispersed mammary cells and plasma membrane-enriched fractions obtained from mammary glands of midpregnant rabbits bound 125I-labeled SC in a saturable time- and temperature-dependent process. The association rate followed a second order reversible reaction (k+1 approximately equal to 2.7 x 10(6) M-1 min-1 at 4 degrees C) and equilibrium was reached in about 4 h at 4 degrees C. The dissociation rate for membranes was first order (k-1 approximately equal to 1.7 x 10(-2) min-1 at 4 degrees C), whereas displacement from cells was incomplete. The apparent affinity constant was similar for membranes and cells (Ka approximately equal to 5 x 10(8) M-1) with one class of binding sites. The number of binding sites varied from one animal to another (260 to 7,000 sites/mammary cell) in relation to endogenous occupancy by SC, which was assessed by immunocytochemistry and complement-mediated cytotoxicity. Rabbit liver and heart membranes did not bind SC, and serum proteins present in rabbit milk failed to interact with mammary cells or membranes. Mammary membranes or cells and liver membranes bound 125I-labeled IgA dimer in a saturable, reversible time- and temperature-dependent process. Association and dissociation rate constants at 4 degrees C (k+1 approximately equal to 5 x 10(6) M-1 min-1 and k-1 approximately equal to 5 x 10(-3) min-1, respectively) and the apparent affinity constant (Ka approximately equal to 10(9) M-1) were similar for liver and mammary membranes; these parameters differed, however, from those reported for free SC-IgA dimer interaction. The binding capacity of membranes for IgA dimer was directly related to the amount of free SC bound to membranes. Interaction of IgA dimer with mammary or liver membranes or cells was abrogated by excess of free SC and was prevented by preincubation of membranes or cells with Fab antibody fragments directed against SC. These data indicate that the first step in the translocation process of polymeric immunoglobulins across epithelia consists of binding of SC to the surface of epithelial cells which in turn acts as a receptor for the specific uptake of IgA dimer.

Unit gravity sedimentation separation of cells comprising the caput epididymidis of the rat.

Cells from the rat caput epididymidis were separated by unit gravity sedimentation. Purest 12-ml fractions contained 88-95% basal cells or 64-76% principal cells. Ultrastructure of separated cells was similar to that of cells in intact tissue. Viability of separated cells was excellent as determined by dye exclusion tests and cellular ATP content. By combining fractions pools containing 4.0 +/- 0.9 X 10(6) cells (86 +/- 8% basal cells) and 1.4 +/- 0.4 X 10(6) cells (56 +/- 7% principal cells) were obtained. Thus, studies on the function of basal and principal cells from the rat caput epididymidis should be possible.

Inhibition of mobility of cell-surface receptors by factors which mediate specific cell-cell interactions.

PREVIOUS studies on embryonic chick neural retina cells1 have demonstrated that cells in intact tissues or single cells prepared by mechanical dispersion have relatively little ability to redistribute their surface receptors into caps. In contrast, cells prepared by trypsinisation and dissociation of intact tissues were found to exhibit lateral redistribution of surface receptors even in the absence of a multivalent ligand. After 4 h culture, however, cells dissociated by trypsinisation have lost the capability for both spontaneus and lectin-induced rearrangements of surface components. Mobility of cell-surface receptors thus seems to be related to, or determined by, trypsin-labile components at the cell surface. One kind of component which seems to be trypsin labile is also important in intercellular recognition and adhesion2,3. We now report that the binding of tissue-type specific cell-surface ligands which seem to mediate recognition and adhesion causes inhibition of directed rearrangement of plant lectin receptors in the plane of the membrane.