Quantitative 125J-Autoradiographie einzelner Zellen

Zusammenfassung Das nach der Energie seiner -Zerfälle zwischen Tritium und Kohlenstoff-14 liegende Isotop ¹²⁵J wurde auf seine Eignung zur quantitativen Autoradiographie geprüft. Absorption und Geometrie-Faktoren der radioaktiven Strahlung wurden untersucht. Hieraus ließen sich geeignete Meßbedingungen entwickeln. Durch gleichzeitige Exposition von radioaktiven Referenzquellen können absolute Radioaktivitätsmengen ermittelt werden. Als Referenzquellen sind membranmarkierte Standardzellen geeignet, die den physikalischen Eigenschaften des Isotopes Rechnung tragen. Hierzu wurden enzymatisch radiojodinierte Schaferythrozyten auf ihre Eignung geprüft. Die absolute Zahl von antigenen Stoffen auf den Oberflächen einzelner Zellen erhält man, wenn man die spezifische Aktivität der markierten Antikörpermoleküle bestimmt und die Silberkorndichte der zu untersuchenden Zellen und der Standardzellen mißt. Die Radioaktivität pro Standardzelle wird in herkömmlicher Weise ermittelt.Die neue Methode wurde zur Quantifizierung membrangebundener Immunglobulinmoleküle vom IgG-Typ auf einzelnen menschlichen Lymphozyten angewendet. Hierbei ist die Ermittlung einer immunologischen Sättigung des markierten Antikörpers wesentlich. Auf Lymphozyten von Normalpersonen und von chronisch-lymphatischen Leukämiepatienten konnten sehr unterschiedliche absolute Immunglobulinmengen bestimmt werden.

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Quantitative ¹²⁵I-autoradiography of individual cells

Summary Iodine 125, an emitter of -radiation with an energy lying between that of tritium and carbon-14, is investigated for its applicability in quantitative autoradiography. Absorption and geometric factors of radiation are elucidated. From this, appropriate measuring conditions are derived. The simultaneous exposure of radioactive standard sources permits the evaluation of absolute amounts of radioactivity. Standard cells with labelled membranes are a suitable source of reference taking into account the physical properties of the isotope. Sheep red blood cells are examined for their suitability as standard cells after enzymatic radioiodination. The absolute number of antigenic substances on the surface of single cells is obtained by determining the specific activity of the labelled antibody molecules, and by measuring the silver grain densities of the cells under investigation and of the standard cells. The radioactivity per standard cell can be assessed by conventional procedures.The new method is applied to the quantification of membrane-bound immunoglobulin molecules of the IgG-type on single human lymphocytes. The determination of an immunologic saturation of the labelled antibody is essential for this purpose. On the lymphocytes of a normal person and of a patient with chronic lymphatic leukaemia quite different amounts of immunoglobulins have been evaluated.

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Assoziation mit EURATOM 031-64 I BIAD.     

Direction of DNA entry in competent cells of Bacillus subtilis

Direction of DNA entry in Bacillus subtilis competent cells was studied using molecules in which only one of the two strands was radioactively labelled. The label was either distributed homogeneously or was localized in a small region of the strand, in the centre or at one of the ends. Regardless of the distribution and the position of the label, similar amounts of radioactivity were taken up by the cells exposed to the labelled molecules. This suggests that DNA enters B. subtilis either by two different uptake systems having opposite polarities, or by a single non-polar system.     

Fc receptors for IgE (Fc epsilon R) on human lymphoid cells: inducible expression of Fc epsilon RII (CD23) on lymphocytes and detection by monoclonal anti-Fc epsilon RII antibody

The studies presented herein describe (1) a sensitive, quantitative, and objective assay for detecting cell membrane-bound form of Fc receptors for IgE displayed on human lymphoid cells based on measuring unlabeled Fc epsilon R-bound IgE by a solid-phase RIA of cell lysate fluids; (2) the development and characterization of an IgM monoclonal antibody, termed 7E4, which is specific for human lymphocyte Fc epsilon RII (CD23) molecules; and (3) a system for reproducibly inducing de novo synthesis and expression of Fc epsilon RII proteins on human lymphocytes following exposure to the mitogenic lectin, pokeweed mitogen. The Fc epsilon RII molecules induced by exposure to PWM were proven to be present on lymphocytes, and not on other cell types in several ways, including (1) documenting sensitivity of such proteins to both acid pH and trypsin treatment, the latter manipulation being ineffective in removing Fc epsilon RII molecules on basophils and mast cells; (2) demonstrating specific reactivity of the expressed Fc epsilon RII molecules with the 7E4 monoclonal antibody, which is specific for human lymphocyte Fc epsilon RII molecules and does not react with Fc epsilon R molecules on other cell types; and (3) observing the required concomitant presence of both T and B lymphocytes during the induction process and proving that the induced Fc epsilon R+ cells are indeed B cells of the Leu-12+ phenotype by fluorescence analysis. The ability to induce expression of Fc epsilon RII molecules on human lymphocytes exposed to a mitogen such as PWM requires special technical attention to the method of preparation and isolation of human lymphoid cells from peripheral blood. This in vitro system for up-regulating Fc epsilon RII expression on human lymphocytes should provide us with an important new tool to analyze the participation of such cells in the regulatory mechanisms controlling the human IgE antibody system.     

Distribution of radioactivity in myelin lipids following subcutaneous injection of [14C]stearate

Blood fatty acids are an important parameter for the synthesis of brain myelin as exogenous stearic acid is needed: after subcutaneous injection to 18-day-old mice this labelled stearic acid is transported into brain myelin and incorporated into its lipids. However the acid is partly metabolized in the brain by elongation (thus providing very long chain fatty acids, mainly lignoceric acid) or by degradation to acetate units (utilized for synthesis of medium chain fatty acids as palmitic acid, and cholesterol). These metabolites are further incorporated into myelin lipids. The myelin lipid radioactivity increases up to 3 days; most of the activity is found in phospholipids; their fatty acids are labelled in saturated as well as in polyunsaturated homologues but sphingolipids, especially cerebrosides, contain also large amounts of radioactivity (which is mainly found in very long chain fatty acids, almost all in lignoceric acid). The occurrence of unesterified fatty acids must be pointed out, these molecules unlike other lipids, are found in constant amount (expressed in radioactivity per mg myelin lipid).     

Detection and measurement of a single blood cell surface antigen by thermal lens microscopy

A highly sensitive method for detection of antigens on the surface of a single blood cell using thermal lens microscopy is described. Colloidal gold, coated with antibody, was used to stain membrane antigens of leukocytes. Human leukocyte antigens on the lymphocytes and mononuclear leukocytes were observed by new thermal lens microscopy, which involves spectrometry using a laser-induced thermal-lens effect. Antigens of HLA-A, -B, and -C loci on the lymphocytes were identified and quantitated using a single cell. The image of HLA-A, -B, and -C antigen distribution on a mononuclear leukocyte was obtained. Our laser microscope, newly devised for measuring convex surface cells, is a powerful analytical tool for detecting and quantitating localized antigens in a single cell and/or cell-surface-associated molecules.     

Anabolism versus catabolism of [5-3H]uridine and its relationship to ribonucleic acid labelling in mouse liver after partial hepatectomy

The balance between anabolism and catabolism of [5-(3)H]uridine was studied in the mouse after partial hepatectomy. Labelling of RNA and UDP-glucose was determined and evaluated in relation to changes in the specific radioactivity of UTP. The amounts of labelled catabolic products of uridine were increased several-fold in liver and blood after partial hepatectomy. The specific radioactivity of RNA decreased to about 60% of the control value at 6h and was in the same range as that of control liver at 24h after operation. Decreased labelling of RNA and UDP-glucose was attributable to decreased specific radioactivity of UTP. No changes in the size of the UTP pool or in the balance between uridine anabolism and catabolism were found that could explain the decreased specific radioactivity of UTP. Rather, the alterations in the labelling of this metabolite induced by the partial hepatectomy may be related to decreased phosphorylating capacity in the liver cells and/or dilution of the labelled precursor in an expanded uridine pool. The enhanced amounts of uridine catabolic products in liver and blood were probably a consequence of accumulation and altered incorporation of the metabolites from the blood into the liver cells. Despite the increased amounts of labelled catabolic products and the decreased labelling of RNA, the results reported here actually suggest decreased uridine catabolism and slightly increased RNA synthesis in mouse liver after partial hepatectomy. The results stress the importance of proper controls in determination of nucleic acid synthesis and in metabolic studies by use of labelled precursors.     

Rates of degradation and synthesis of glycosidases de novo during growth and differentiation of Dictyostelium discoideum.

1. Injection of a purified preparation of beta-N-acetylglucosaminidase from the spent growth medium of myxamoebae of Dictyostelium discoideum into rabbits gave rise to an antibody preparation containing both anti-alpha-glucosidase and anti-beta-acetylglucosaminidase activities. 2. These two activities were shown to reside in different immunoglobulin molecules and it was concluded that the beta-N-acetylglucosaminidase preparation contained trace amounts of highly antigenic alpha-glucosidase. 3. A single precipitin band having beta-N-acetylglucosaminidase activity was formed in Ouchterlony plates when this antibody preparation was tested against extracts obtained from differentiated cells or from myxamoebae grown either axenically or on bacteria. 4. The antibody preparation was used to show that both beta-N-acetylglucosaminidase and alpha-glucosidase molecules are synthesized de novo from isotopically labelled amino acids during both the growth and differentiation phases of the life cycle and to show that neither of these proteins is significantly degraded during the growth phase or during the first 9h of differentiation. 5. The rates of accumulation of these assayable enzyme activities are thus equal to their rates of synthesis during growth and early differentiation. 6. The factors regulating cellular enzyme activity during the life cycle of D. discoideum are discussed.     

Formation of nascent DNA molecules during inhibition of replicon initiation in mammalian cells.

X-irradiation of mammalian cells with moderate doses (100-1000 rads) inhibits the initiation of DNA replicons. This inhibition is observed as depressed amounts of radioactivity at low molecular weights when the DNA from the cells is analysed by velocity sedimentation in alkaline sucrose gradients at 30 min after irradiation. There is no detectable effect on chain elongation and joining of those molecules that do initiate replication; this is indicated by the presence of the same amounts of radioactivity in nascent DNA molecules of high molecular weights from control and irradiated cells. The labeling of DNA molecules that initiated replication before irradiation continues unhindered for more than 60 min after irradiation, which is observed as peaks of radioactivity at high S values in alkaline sucrose gradients from irradiated cells. These data indicate that DNA replication in mammalian cells proceeds by continuous joining of nascent molecules that initiate almost simultaneously at origins at various distances from one another. Some of the interorigin distances are much greater than others, implying that large replicons make up a significant component of mammalian DNA.     

A monoclonal antibody identifying a T-cell marker in the horse

A cell surface molecule of equine T lymphocytes was identified and characterized using a mouse monoclonal antibody, HT23A. The molecule was detected on all T cells but not on other cells in peripheral blood, with the possible exception of a small subpopulation (about 5%) of B cells, as assessed by indirect immunofluorescence and flow cytometry. HT23A labelled T cell areas of horse lymph nodes and spleen when used in an indirect immunoperoxidase assay on frozen sections. Macrophages and neutrophils were not labelled by the antibody nor were frozen sections of horse liver, kidney, or brain. HT23A precipitated a molecule of approximately 69 kDa from 125Iodine labelled horse lymphocytes.     

Automated single-cell analysis of aryl hydrocarbon hydroxylase in human lymphocytes

A method of rapidly analyzing hydroxybenzo (a) pyrene in single human lymphocytes with simultaneous sorting is reported. Cells with various levels of hydroxybenzo (a) pyrene were characterized and sorted by means of a Fluorescence Activated Cell Sorter into three groups with low, intermediate and high amounts of benzo (a) pyrene metabolite. The mean amount per cell was calculated after measuring the fluorescence of the cell suspensions in a fluorometer. Hydroxybenzo (a) pyrene may be detected and quantitated in individual cells at a level of sensitivity less than 10⁶ molecules per cell. The sorting capability allows for retrieval of precisely selected samples of cells for further studies of similar or other parameters.     

ESTIMATION OF THE MIGRATION OF THORACIC DUCT LYMPHOCYTES TO NON-LYMPHOID TISSUES

The distribution of radioisotopes in tissues was measured following i.v. injection of labelled thoracic duct lymphocytes into syngeneic rats. The rate of elution of an isotope from the labelled cells and the subsequent fate of the eluted isotope were shown to be the most important factors limiting the usefulness of such isotopes for measuring cell localization particularly in non-lymphoid tissues. Comparison of labelling procedures using [³H] and [¹⁴C]uridine, [³H] and [¹⁴C]leucine, [⁷⁵Se]-L-selenomethionine, [^99mTc]sodium pertechnetate and [⁵¹Cr]sodium chromate in vitro and [³H]thymidine in vivo showed that ⁵¹Cr had the fewest disadvantages in the present context. Using ^5ICr-labelled cells, the radioactivity was measured in a wide range of non-lymphoid tissues, and estimates of cell traffic were obtained. In skin, for example, the results indicate a cell flux in the range of 10⁴-10⁵ lymphocytes/gm/hr. Evidence is presented which suggests that the early substantial localization of labelled cells in the lung is not an artefact due to sequestration or embolization of traumatized cells but probably reflects a slow intravascular transit time through this capillary bed. The primary lymphoid organs, thymus and bone marrow were shown to include a subpopulation of lymphocytes which belong to the recirculating pool. The thymus always contained a greater concentration of radioactivity at 24 hr than all non-lymphoid tissues except liver and kidney (approx. 0-1% of the recirculating lymphocyte pool) and the bone marrow was capable of temporarily accepting a substantial proportion (approx. 25%) of the injected cells.     

Different metabolic recycling of the lipid components of exogenous sulphatide in human fibroblasts.

Cultured human fibroblasts were fed with two differently labelled sulphatide molecules [one labelled on C-3 of the sphingosine (Sph) moiety [( Sph-3H]sulphatide), the second on C-1 of stearic acid [( stearoyl-14C]sulphatide)], and the intracellular metabolic fate of radioactivity was monitored. Incorporated radioactivity was almost all recovered in the total lipid extract, regardless of the labelling position of the added sulphatide; however, large differences in the level of incorporation occurred among labelled glycosphingolipids. For example, sphingomyelin was present as the major radiolabelled lipid after [Sph-3H]-sulphatide incubation, but was detectable only in trace amounts after [stearoyl-14C]sulphatide administration; in the latter case the radioactivity was located predominantly in glycerophospholipids. From this finding it can be inferred that the free long-chain base (sphingosine) that originates from lysosomal catabolism of sulphatide is mainly, and quite specifically, utilized for sphingomyelin biosynthesis, whereas the ceramide moiety is not; conversely the fatty acid released from ceramide is non-specifically re-utilized for phospholipid biosynthesis.     

Calreticulin is expressed on the cell surface of activated human peripheral blood T lymphocytes in association with major histocompatibility complex class I molecules.

Calreticulin is an endoplasmic reticulum resident molecule known to be involved in the folding and assembly of major histocompatibility complex (MHC) class I molecules. In the present study, expression of calreticulin was analyzed in human peripheral blood T lymphocytes. Pulse-chase experiments in [35S]methionine-labeled T cell blasts showed that calreticulin was associated with several proteins in the endoplasmic reticulum and suggested that it was expressed at the cell surface. Indeed, the 60-kDa calreticulin was labeled by cell surface biotinylation and precipitated from the surface of activated T cells together with a protein with an apparent molecular mass of 46 kDa. Cell surface expression of calreticulin by activated T lymphocytes was further confirmed by immunofluorescence and flow cytometry, studies that showed that both CD8+ and CD4+ T cells expressed calreticulin in the plasma membrane. Low amounts of cell surface calreticulin were detected in resting T lymphocytes. By sequential immunoprecipitation using the conformation independent monoclonal antibody HC-10, we provided evidence that the cell surface 46-kDa protein co-precipitated with calreticulin is unfolded MHC I. These results show for the first time that after T cell activation, significant amounts of calreticulin are expressed on the T cell surface, where they are found in physical association with a pool of beta2-free MHC class I molecules.     

Integration of donor DNA in bacterial conjugation

Conjugation between ¹³C¹⁵N- and ³H-labelled hybrid donors and ¹³C¹⁵N-labelled hybrid recipients of Escherichia coli gives rise to recombinant radioactive DNA of density greater than labelled hybrid. The donor radioactivity is present, in these molecules, in discrete heavy segments covalently attached to the light strand. When light radioactive Hfr cells are mated to heavy F^? cells in light medium, the donor label appears, in DNA extracted from the F^? cells, in labelled hybrid molecules. The radioactivity in these molecules is exclusively in the light strand. The insertion of donor material is thus restricted to a single newly formed strand of the recipient DNA and double-strand integrations do not occur. A temperature-sensitive recipient containing the dna B mutation ts₄₃ accumulates single-stranded Hfr DNA if mating is carried out at the nonpermissive temperature. The formation of a complementary strand in the recipient does not, therefore, appear to be necessary for continued transfer of Hfr DNA.     

A novel spectrofluorometric technique for specific biocompatibility testing of implantable materials by cell culture. Report on use for multiparameter analysis of human osteoblasts cultured on commercially pure titanium and hydroxyapatite

The authors describe a novel spectrofluorometric technique based on double-labelled fluorescence imaging using immunoconjugates labelled with fluorochromes. Following isolation and characterization, cells are seeded on the surface of disks of the material(s) to be tested. After application of a primary antibody and an antibody bearing a fluorochrome, the signal emitted by the molecules in the extracellular matrix on the surface of the test disks is measured by spectrofluorimetry. Measurement is thus independent of the surface characteristics of the test material. Measured values are compared with pre-established standard curves. This technique facilitates determination of the characteristic molecules expressed by a given cell type,thus allowing accurate evaluation of the response of pertinent biological samples to implantable biomaterials. This revised version was published online in July 2006 with corrections to the Cover Date.     

Immunohistochemical identification of T- and B-lymphocytes delineated by the unlabelled antibody enzyme method

Summary T-lymphocytes and B-lymphocytes are identified in tissue sections of human tonsils by applying the unlabelled antibody enzyme method. The epithelium of the tonsils contains a majority of immunoglobulin-positive cells and fewer T-lymphocytes. In the subepithelial zones, areas composed of B-cells predominate, however, regions containing T-lymphocytes are also present. The latter are mainly arranged in the lamina propria around high-endothelial venules and often include plasma cells containing immunoglobulin. Follicles containing germinal centres display a complex structure which changes during development. The lymphocytic cap consists of densely packed lymphocytes, labelled heavily by anti-IgM and anti-IGD, and of individual T-lymphocytes. Germinal centres show a framework of immunoglobulin-positive dendritic reticular cells; they contain some heavily labelled lymphoid cells and several cells weakly labelled by anti-IgM and anti-IgA, as well as a small number of T-lymphocytes. Furthermore, the total areas of T- and B-lymphocytes measured by planimetry may differ considerably between different tonsils. Especially total areas of germinal centres vary to a great extent. The quantitative data on amounts of T- and B-cells achieved by planimetry are comparable to those reported in cellular suspensions of tonsils.     

Direct measurement of femtogram amounts of DNA in cells and chloroplasts by quantitative microspectrofluorometry

Absolute DNA amounts of individual chloroplasts were determined by measuring the fluorescence intensity of chloroplasts stained with 4',6-diamidino-2-phenylindole (DAPI) relative to that of the bacterium Pediococcus damnosus (cerevisiae) smeared on the same slide. An absolute DNA content of 7.7 X 10(15) g for a standard P. damnosus cell type was calculated by comparing the relative fluorescence values and frequency of each stage of cellular development in a culture to the average DNA content of all cell types determined by chemical methods. Chlorophyll was extracted from the chloroplasts during fixation so that chlorophyll autofluorescence was not present when DAPI fluorescence was measured. Absolute amounts of DNA could then be determined for single chloroplasts, either within cells that were individually selected from a mixed cell population or in small preparations of isolated chloroplasts. The DNA amounts of chloroplasts from mesophyll cells determined in this way were similar to the values previously determined by bulk averaging methods. Chloroplast DNA amounts from different cell types of the leaf could be measured by microspectrofluorometry, and it was found that chloroplasts from spinach epidermal cells contained about half as much DNA as chloroplasts from adjacent mesophyll cells.     

Cell surface glycoproteins of CHO cells. I. Internalization and rapid recycling

The major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4 degrees C with the membrane-impermeant reagent, trinitrobenzenesulfonate (TNBS). Immunoprecipitation and subsequent two-dimensional, sodium-dodecyl sulfate, polyacrylamide gel electrophoresis (SDS-PAGE) of proteins from derivatized cells that had been labelled previously with [3H]D-glucosamine or [3H]L-leucine showed that TNBS reacted with most of the high molecular weight (HMW) acidic glycoproteins that became labelled with iodine by the lactoperoxidase technique and that bind the lectin, wheat germ agglutinin (WGA). After warming the cells to allow endocytosis to proceed, molecules haptenized with trinitrophenol (TNP) groups were followed radiochemically by means of [125I]anti-DNP antibodies. The half-life for internalization of proteins tagged with either [125I]anti-DNP IgG or Fab averaged about 5 min. A similar result was obtained when a monoclonal antibody directed against a single plasma membrane glycoprotein was used, or when the rate of surface loss of TNP groups unoccupied by antibodies was measured. Within 15 min at 37 degrees C, a steady-state between surface and cytoplasmic label was reached, with about 65% of the hapten located internally. Recycling of internalized TNP groups back to the cell surface also occurred rapidly (t 1/2 approximately 5 min). Most of the intracellular radioactivity was associated with a membrane fraction of density similar to that of the plasma membrane. Over a 4-h period, there was no significant entry of labeled molecules into lysosomes. By contrast, the fluid-phase marker, horseradish peroxidase, became associated with the lysosomes within 1 h. Our results are consistent with the view that the majority of plasma membrane glycoproteins are continuously being internalized and recycled at a high rate.     

The expression of melanosomal matrix protein in the transdifferentiation of pigmented epithelial cells into lens cells

A monoclonal antibody (MC/1) was constructed against melanosomes purified from the chicken pigmented epithelial cells (PECs) in order to characterize the differentiative phenotypes of PEC in the process of transdifferentiation into lens cells. Immunofluorescent studies revealed that MC/1 antibody specifically stains both retinal PECs in the eye and melanocytes in the skin, of chicken embryos. Immunoelectron microscopy showed that the antigen molecules are located on the peripheral region of the melanosomal matrix. A single protein band with an apparent molecular weight of 115,000 was labelled by MC/1 in Western blotting. The 115 kDa polypeptide identified by MC/1 is considered to be a member of the melanosomal matrix proteins. The maintenance of specificity of pigment cell nature is followed in the system of transdifferentiation of PEC into lens in vitro, utilizing 115 kDa protein as a marker. In the dedifferentiated PECs, this protein was undetectable.