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Biomedical subjects

G Setterfield

Publications and source records attributed to G Setterfield.

At least 19 recordsLinked to original sources
PubMed1988

Changes in structure and protein composition of bovine lymphocyte nuclear matrix during concanavalin-A-induced mitogenesis.

A major component of nuclear change in concanavalin-A-stimulated bovine lymphocytes is a severalfold increase in interchromatinic volume, which coincides with nuclear swelling and extensive structural remodelling. Large-scale ultrastructural changes in isolated nuclei and nuclear matrices (NM) reflect those occurring within nuclei in situ during mitogenesis. While nonchromatinic nuclear material embedded within nuclease- and salt-extracted whole cells closely resembled in situ interchromatinic matrices, large NM isolated in solution shrank after chromatin was extracted. Numerous perinuclear filaments persisted throughout NM isolation and cytoskeletal proteins were identified in two-dimensional (2-D) gels of such preparations. Taken together these data indicated that the lymphocyte cytoskeleton is likely continuous with the nuclear matrix and could play a role in maintaining nuclear organization. A wide range of lymphocyte NM proteins were resolved in 2-D gels. Significant changes in protein composition coincided with nuclear structural remodelling. Lamin B was prominent at each stage of nuclear development, whereas lamins A and C were only found in stimulated lymphocyte matrices. Lymphoblast NM contained more large basic proteins. Progressively increasing polypeptide complexity of these NM arose by de novo protein synthesis and posttranslational modifications throughout concanavalin A stimulation. NM from stimulated lymphocytes also contained more ribonucleoproteins, possibly indicating the presence of significant amounts of transcriptional material.

Animals↗
PubMed1985

Non-Hodgkin's lymphoma classification: ultrastructural morphometric studies for the quantification of nuclear compartments in situ.

The condensed chromatin distribution in the nuclei of lymphocytes in non-Hodgkin's lymphoma (NHL) is a key element, along with nuclear size and shape, in the classification of this disease for therapeutic and prognostic purposes. This report describes the ultrastructural comparative quantification of the condensed chromatin and the interchromatinic (nuclear matrix or euchromatin) region in the nuclei of mitogen-stimulated human peripheral T lymphocytes and mouse spleen B lymphocytes, human germinal center lymphocytes, and lymphocytes in ten cases of NHL of a variety of subtypes. The sequential morphologic nuclear changes induced in lymphocytes by mitogens are reflected in human germinal center lymphocyte populations. The common features include the changes in the distribution and volume of condensed chromatin aggregates, as well as the fact that the major increments in nuclear volume during lymphocyte transformation result from increases in the volume of the interchromatinic region. In all subtypes of NHL analyzed morphometrically, subpopulations of lymphocytes were identified in which mean nuclear, condensed chromatin, and interchromatinic volumes were more or less equivalent to those of normal lymphocyte subsets in germinal centers in reactive hyperplasia. However, in NHL the abnormal cytologic characteristics of the nucleus result, at least in part, from a complex interplay of condensed chromatin distribution and amount, and the size of the interchromatinic region. Further complexity is introduced by the fact that in NHL these two nuclear compartments can independently be normal, increased, or reduced in size. Morphometric quantification of lymphocytes in NHL indicates that the interchromatinic (matrix) region of the nucleus is the key element in establishing the nuclear volume of neoplastic lymphocytes. The structural and functional, ribonucleoprotein interchromatinic region of the nucleus was visualized in normal and neoplastic lymphocytes by regressive uranyl-EDTA staining. Quantitative morphometric analysis indicates that the cytologic appearance of neoplastic lymphocytes, even within subtypes of NHL, is heterogeneous and that condensed chromatin quantity and distribution may be more critical than nuclear size in distinguishing between certain subtypes of NHL. Improvements in the classification of NHL will occur only with understanding of the alterations in the biologic mechanisms controlling gross nuclear organization and the morphologic events of the various differentiation pathways available to antigen-stimulated lymphocytes.

Animals↗
PubMed1985

Evaluation of techniques for immunofluorescent staining of microtubules in cultured plant cells.

Various modifications to the immunofluorescent labeling procedures for microtubules in plant cells have been compared using cell cultures of Vicia hajastana Grossh. Using serial section electron microscopic reconstructions as a reference, we have chosen as our standard procedure a method that maximizes both the preservation of the cytoskeleton and the proportion of cells staining, while minimizing the degree of nonspecific staining. The critical steps of the procedure include stabilization of the cytoskeleton, cell wall permeabilization, and cell extraction. To maintain structural integrity during the procedure, it is necessary to stabilize the cytoskeleton with paraformaldehyde. To facilitate antibody penetration into the cell, it was necessary that the walls be made permeable via partial enzymatic digestion. Detergent extraction of cells increased the proportion of cells staining and decreased the level of nonspecific binding of the antibodies. The procedures detailed in this article provide a good starting point for the application of immunofluorescent labeling techniques to other plant systems.

Fluorescent Antibody Technique↗
PubMed1984

Changes in distribution of nuclear matrix antigens during the mitotic cell cycle.

We examined the distribution of nonlamin nuclear matrix antigens during the mitotic cell cycle in mouse 3T3 fibroblasts. Four monoclonal antibodies produced against isolated nuclear matrices were used to characterize antigens by the immunoblotting of isolated nuclear matrix preparations, and were used to localize the antigens by indirect immunofluorescence. For comparison, lamins and histones were localized using human autoimmune antibodies. At interphase, the monoclonal antibodies recognized non-nucleolar and nonheterochromatin nuclear components. Antibody P1 stained the nuclear periphery homogeneously, with some small invaginations toward the interior of the nucleus. Antibody I1 detected an antigen distributed as fine granules throughout the nuclear interior. Monoclonals PI1 and PI2 stained both the nuclear periphery and interior, with some characteristic differences. During mitosis, P1 and I1 were chromosome-associated, whereas PI1 and PI2 dispersed in the cytoplasm. Antibody P1 heavily stained the periphery of the chromosome mass, and we suggest that the antigen may play a role in maintaining interphase and mitotic chromosome order. With antibody I1, bright granules were distributed along the chromosomes and there was also some diffuse internal staining. The antigen to I1 may be involved in chromatin/chromosome higher-order organization throughout the cell cycle. Antibodies PI1 and PI2 were redistributed independently during prophase, and dispersed into the cytoplasm during prometaphase. Antibody PI2 also detected antigen associated with the spindle poles.

Animals↗
PubMed1984

Purine and pyrimidine analogues irreversibly prevent passage of lymphocytes from the G1 to the S phase of the cell cycle.

Six-hour pulses of the purine analogue 8-azaguanine (8-AG) and the pyrimidine analogue 5-fluorouracil (5-FU) produced a novel irreversible effect on mouse and human lymphocytes. Cells treated with these analogues early during culture with concanavalin A and then washed in presence of excess natural base could pass normally through the various stages of blast formation (e.g., increased K+ transport, increase in nuclear and cytoplasmic volume, disaggregation of chromatin), but showed a severe inhibition of DNA synthesis when this was measured by [3H]thymidine incorporation at 48 h of culture; this was true irrespective of whether the 6-h pulse with analogue occurred at 0, 12, or 24 h of culture in the presence of mitogen. The analogue 6-mercaptopurine, which strongly inhibited DNA and RNA synthesis while present in the medium, had no irreversible effects, unlike 5-FU and 8-AG. The persistence of the effects of 5-FU in presence of excess thymidine in the medium suggested that inactivation of thymidylate synthetase was not responsible for the inhibition observed here. The effect was expressed in the presence or absence of protein synthesis; therefore, the observed inhibition of proliferation was not due to synthesis of a toxic protein, but to an effect on the formation or function of the DNA synthesizing system and (or) on its template, thus preventing the cells from passing from the G1 to the S phase of the cell cycle.

Animals↗
PubMed1983

Changes in structure and composition of lymphocyte nuclei during mitogenic stimulation.

Nuclei of lymphocytes stimulated in vitro with concanavalin A (Con A) were classified into three morphotypes: I--unstimulated; II--partially stimulated; III--fully stimulated, lymphoblastic nuclei. During the Con A-induced change from morphotype I to III nuclear volume increased up to sixfold, due to a near 10-fold increase in the interchromatinic region. At the same time, condensed chromatin rose in volume by only about 1.5-fold and became disaggregated in to small clumps. Regressive EDTA-uranyl staining demonstrated a large increase in interchromatinic fibrillar material in morphotypes II and III. Nuclear matrices isolated from stimulated murine lymphocytes showed structures comparable to the interchromatinic region of the morphotypes. The Con A-stimulated change in nuclear structure preceded onset of DNA replication and was unaffected by hydroxyurea or cytosine arabinoside. Cycloheximide blocked the structural change, even when given 20 hr after Con A. Autoradiography after [3H]leucine showed incorporation of label in the interchromatinic region of morphotype II and III nuclei, much of which remained stable during a 48-hr chase period. Nuclear structural activation was inhibited by alpha-amanitin but a significant stable nuclear RNA fraction was not detected. We conclude that an important event in lymphocyte activation is extensive synthesis of stable proteinaceous interchromatinic matrix which may be involved in chromatin remodeling and DNA replication and/or transcription.

Animals↗
PubMed1983

Nuclear bodies in mouse splenic lymphocytes: II - Cytochemistry and autoradiography during stimulation by concanavalin A.

Nuclear bodies (NB) are poorly understood nucleoplasmic structures frequently observed in many animal cell types. Murine lymphocytes mitogenically stimulated with concanavalin A contain 5 types of NB. In order to examine the origin and function of the NB we have carried out cell fractionation and have performed cytochemistry, immunocytochemistry and autoradiography at the electron microscope level. Regressive staining for RNP showed that simple NB (types I and II) and the shells of complex NB (types III, IVa and V) consist of a non-chromatinic fibrillar material which is most likely proteinaceous. Projections of this material from the NB surface appear to link the NB to nucleoplasmic fibrogranular elements. The fibrillar, filamentous and granular components of the cores of complex NB were largely RNP and in some instances closely resembled fibrogranular areas in the nucleoplasm. Chromatin masses were seen occasionally in the cores. All NB types remained unstained after nucleolus-specific silver nitrate staining, and were also not stained by a centromere-specific antibody. Autoradiography was performed along long-term labelling with 3H-uridine, 3H-thymidine or 3H-leucine. The NB were not labelled by any of the precursors, indicating that macromolecular synthesis does not play a major role in NB evolution in these cells. By fractionation, we showed that NB co-isolated with the nuclear matrix, and were linked to the fibrogranular nuclear matrix component by projections from the surface of the NB. A possible role of NB in RNA processing is discussed.

Animals↗
PubMed1983

Nuclear morphology and morphometry of B-lymphocyte transformation. Implications for follicular center cell lymphomas.

One of the major tenets of current non-Hodgkin's lymphoma classifications is the relationship of morphologic subtypes to stages in the sequence of normal B-lymphocyte transformation occurring in the germinal follicle. To test this hypothesis, quantitative morphometric image analysis was carried out on in vivo and in vitro samples of mouse splenic lymphocytes in which transformation was induced by bacterial lipopolysaccharide (LPS), a specific B-cell mitogen. The results were compared with a similar analysis of germinal center lymphocyte populations of normal human spleen. In the in vivo mouse model, initial stages of B-cell transformation were detectable as early as 4 hours after LPS injection, and the process was essentially fully developed by 48-72 hours. Quantitative evaluation revealed that the majority of nuclear profiles were nearly spherical or only slightly eccentric and that no major alteration in nuclear contour occurred during any phase of the progressive increase in nuclear size following mitogen-induced lymphocyte transformation. In fact, in this system, B lymphocytes with a nuclear profile cleft of greater than or equal to 0.4 mu accounted for only 3% of the combined unstimulated and LPS-activated population assessed (N = 9936). This compared with normal human spleen, in which 16% of germinal center lymphocyte populations had similarly cleft nuclear profiles. Sequential alterations in the organization of condensed chromatin occurred concomitant with gradual nuclear enlargement during mitogen-induced mouse spleen lymphocyte transformation. A comparative morphologic and morphometric assessment of nuclear profiles of lymphocyte populations in germinal centers of normal human spleen provides indirect evidence for a similar pattern of nuclear alterations in human B lymphocytes. Autoradiographic data obtained from LPS-activated mouse splenic lymphocytes indicate that nuclear morphologic aspects of the transformation process can occur entirely within the G1 phase of the cell cycle. The results of this study suggest that subtypes of non-Hodgkin's lymphoma largely composed of neoplastic lymphocytes with extensively convoluted or cleft nuclei do not reflect a morphologic stage in the transformation of normal lymphocytes. In addition, the heterogeneous nuclear forms of follicular center cell lymphocytes would appear to result from parallel transformation processes involving cleaved and noncleaved nucleated lymphocytes and not the sequential pathway proposed by Lukes and Collins.

Animals↗
PubMed1983

Organization of microtubules in dividing and elongating cells of Vicia hajastana Grossh. in suspension culture.

Cells of Vicia hajastana Grossh. cultured with 2,4-D showed coupled division and growth and formed multicellular files of small isodiametric cells. In GA without added 2,4-D, the cells stopped dividing and continued elongating for several days. Total growth was the same in both hormone conditions. An immunofluorescent technique was developed to study microtubule (MT) distribution. Cells in GA showed parallel MT arrays oriented transversely to the axis of elongation. In some cells the number of MT per unit length was maintained during growth while other elongating cells showed reduced frequency of MT. Microtubules often appeared as thickened, branched strands, probably as a result of lateral aggregation. In cells grown in 2,4-D some pre-prophase bands of MT were observed. Cells in mitosis lacked cortical MT, and all organized staining was in spindles or phragmoplasts. Interphase cells in 2,4-D showed variable organization of cortical MT ranging from disordered to transversely ordered. Cells in early interphase had disordered MT while larger cells showed order. These observations indicate that MT in cycling cells are continually changing organization, probably accounting for the different distributions observed in interphase cells. On cessation of the mitotic cycle, reorganization of MT stops and transverse arrays of cortical MT are maintained as cells elongate. These processes are similar to those observed in organized tissues; however, cultured cells offer distinct advantages for experimental manipulation and microscopic observation of cytoskeleton.

Cell Division↗
PubMed1982

Immunofluorescent localization of lysine-rich histones in isolated nuclei from adult and embryonic chicken erythrocytes.

Isolated nuclei from adult chicken erythrocytes were stained by indirect immunofluorescence for histones H5 and H1. Nuclei in 0.15 M NaCl stained for H5 showed internuclear variations in intensity of fluorescence from bright to dim. Most individual nuclei were homogeneously stained although some showed a bright rim around a dimmer interior. Treatment of nuclei with Tween 80 in 0.15 or 0.03 M NaCl also gave internuclear variation in intensity. Adult nuclei stained for H1 (in 0.15 or 0.03 M NaCl) showed little internuclear variation; most nuclei stained brightly with a brighter rim. Simultaneous staining of H5 and H1 in the same nuclei confirmed the variable fluorescence of H5 and consistent fluorescence of H1. Most nuclei showed the presence of both histones. Nuclei from embryonic blood cells also showed considerable internuclear variation of H5 fluorescence and less variation with H1 staining. For both histones the proportion of brightly staining nuclei increased with embryonic development. Difficulties in interpreting quantitative variations in immunofluorescence are discussed.

Animals↗
PubMed1981

Nuclear alterations during lymphocyte transformation: relationship to the heterogeneous morphologic presentations of non-Hodgkin's lymphomas.

A current hypothesis related to non-Hodgkin's lymphoma states that the wide variety of cytologic types in this disorder reflects morphologic alterations during different stages (G1, S, and G2) of the cell cycle involved in the blastogenic transformation of normal lymphocytes. In our investigations of biochemical and structural changes during lymphocyte transformation, we have used correlated stereologic morphometric analysis, assessment of chromatin organization, and autoradiography of human peripheral T-lymphocytes labeled with 3H-thymidine and stimulated with concanavalin A. These studies have confirmed that the characteristic increase in nuclear size and disaggregation of condensed chromatin masses precedes and is independent of DNA synthesis. Since the full range of morphologic alterations observed in lymphocyte transformation can occur in the G1 phase of this process, modifications to the above hypothesis are required. Assessment of the nuclear contour index following mitogen stimulation indicates that at least in this in vivo system, there is no cleaved or convoluted phase during the transformation of human peripheral T lymphocytes.

Autoradiography↗
PubMed1980

Relationship between chromatin structure and replication in mouse L-cells.

Mouse L-cells treated with cytosine arabinoside, hydroxyurea, fluorodeoxyuridine, methotrexate, or mitomycin C rapidly cease DNA synthesis and stop dividing. Such inhibition of DNA replication is followed by interruption of formation of lysine- and arginine-containing proteins, including chromatin-bound histones, and by a major reorganization of the heterochromatin of the central nucleoplasm, manifest as disaggregation of large clumps of this condensed chromatin. Morphometric analysis revealed both cell and nuclear enlargement in cells treated with such antimetabolites of DNA replication. These observations are in contrast to those made with WT-4 cells starved of isoleucine or treated with cycloheximide. Isoleucine depletion was associated with inhibition of DNA synthesis and continued increase of cell and nuclear volume, but not with massive disaggregation of heterochromatin. Cycloheximide produced inhibition of DNA synthesis and protoplasmic growth, and also prevented structural reorganization of chromatin. A model is presented which suggests that initiation of chromatin replication is associated with a process, dependent upon de novo protein synthesis, which results in chromatin disaggregation. This can be revealed by inhibition of the correct replication of chromatin DNA and chromatin protein.

Animals↗
PubMed1978

Early origins of definitive erythroid cells in the chick embryo.

The early maturation stages of definitive erythroid cells are observed in the embryonic circulation of the chick yolk sac at 4.5--5 days of incubation. Light and electron microscope observation of the mesoderm of the yold sac membrane indicate that individual presumptive precursors of the definitive-line are present as early as 2 days of incubation and give rise to sequestered populations of immature erythroblasts within sinusoids during the period of 2.5-6 days incubation. Such isolated populations of definitive-line erythroblasts eventually connect with the established capillary circulation of yolk sac membrane but a large proportion of the erythroblasts temporarily remain associated with the endothelium prior to free circulation.

Animals↗
PubMed1978

Structure of interphase nuclei in relation to the cell cycle. Chromatin organization in mouse L cells temperature-sensitive for DNA replication.

Mutant lines of mouse L cells, TS A1S9, and TS C1, show temperature-sensitive (TS) DNA synthesis and cell division when shifted from 34 degrees to 38.5 degrees C. With TS A1S9 the decline in DNA synthesis begins after 6-8 h at 38.5 degrees C and is most marked at about 24 h. Most cells in S, G2, or M at temperature upshift complete one mitosis and accumulate in the subsequent interphase at G1 or early S as a result of expression of a primary defect, failure of elongation of newly made small DNA fragments. Heat inactivation of TS C1 cells is more rapid; they fail to complete the interphase in progress at temperature upshift and accumulate at late S or G2. Inhibition of both cell types is reversible on return to 34 degrees C. Cell and nuclear growth continues during inhibition of replication. Expression of both TS mutations leads to a marked change in gross organization of chromatin as revealed by electron microscopy. Nuclei of wild-type cells at 34 degrees and 38.5 degrees C and mutant cells at 34 degrees C show a range of aggregation of condensed chromatin from small dispersed bodies to large discrete clumps, with the majority in an intermediate state. In TS cells at 38.5 degrees C, condensed chromatin bodies in the central nuclear region become disaggregated into small clumps dispersed through the nucleus. Morphometric estimation of volume of condensed chromatin indicates that this process is not due to complete decondensation of chromatin fibrils, but rather involves dispersal of large condensed chromatin bodies into finer aggregates and loosening of fibrils within the aggregates. The dispersed condition is reversed in nuclei which resume DNA synthesis when TS cells are downshifted from 38.5 degrees to 34 degrees C. The morphological observations are consistent with the hypothesis that condensed chromatin normally undergoes an ordered cycle of transient, localized disaggregation and reaggregation associated with replication. In temperature-inactivated mutants, normal progressive disaggregation presumably occurs, but subsequent lack of chromatin replication prevents reaggregation.

Cell Count↗
PubMed1978

Mutant mouse L-cells: a model for megaloblastic anaemia.

When temperature-sensitive (ts) mutant lines of mouse L-cells, ts AIS9 and ts CI, are shifted from 34C to 38.5C, a rapid inhibition of DNA synthesis and mitosis occurs. During this phase, cell and nuclear growth continues and results in a substantial increase in cell and nuclear volume. Such cellular modifications are also associated with a marked dispersal of the condensed chromatin masses of interphase nuclei, so that after 48-72 h of incubation at 38.5C, nuclear profiles of both ts cell lines bear a striking resemblance to the nuclear features characteristic of megaloblastic anaemia. Despite these marked alterations in nuclear chromatin organization, morphometric analysis indicates that the volume of condensed chromatin does not decrease. Current biochemical, cytological and morphometric data on the two ts lines of mutant mouse L-cells during expression of the mutation, suggest that they might provide a useful model to further elucidate cytological features of megaloblastic anaemia.

Anemia, Macrocytic↗
PubMed1978

Vectorial transport of proteins by membrane-bound ribosomes of nongrowing and growing Jerusalem artichoke tuber cells.

Both nongrowing (water-incubated) and growing (hormonally stimulated) Jerusalem artichoke tuber cells contain membrane-bound (mb) ribosomes. Using a rapid flotation procedure, a membrane fraction was prepared from both types of cells. This fraction was enriched in mb ribosomes, contained NADH cytochrome c reductase activity, had RNA:phospholipid and RNA:protein ratios similar to those reported for rough microsomes from animal tissues, and supported synthesis of preinitiated proteins in vitro. Using puromycin and detergent release, vectorial transport of labelled polypeptides was measured in the in vitro system. Of proteins made by mb ribosomes from nongrowing cells, on 12% remained associated with microsome membranes following chain termination. The comparable figure for proteins from mb ribosomes of growing tissue was 42%. The membrane-associated proteins were preferentially protected from protease digestion. Some possible reasons are suggested for the correlation between cell growth and the association of newly synthesized proteins with microsomes. The role of proteins synthesized by mb ribosomes but not vectorially transported, in both growing and nongrowing cells, is unknown.

Cell Membrane↗