STUDIES ON THE REGULATION OF LYMPHOCYTE PRODUCTION IN THE MURINE THYMUS AND SOME EFFECTS OF A CRUDE THYMUS EXTRACT

Cell proliferation in the murine thymus was studied in vivo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the first 24 hr the mitotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0–8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering Go/Gi cells into DNA synthesis, the inhibition of G₂ efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed.     

CYTODYNAMICS IN THE THYMUS OF YOUNG ADULT MICE:

Cell proliferation and cell loss in the thymic blast cell population were studied in young adult mice by (1) stathmokinetic methods combined with an analysis of the PLMe-curve after a pulse ³H-TdR, and (2) nigrosin-dye exclusion combined with ³H-TdR-autoradiography. It was calculated that about 17% of the blast cells do not progress into mitosis within the period of an average cell cycle. The dye exclusion studies indicated a rate of blast cell death of about 2–5 %/hr. The two methods of assessing blast cell loss (death) support each other very well. In spite of these findings scintillation countings on thymuses removed from 1 to 17 hr after ³H-TdR injection showed fairly constant levels of thymic radioactivity. This suggests a very extensive reutilization of ³H-labelled break-down products from dying blast cells. The very sparse labelling of pyknotic thymocytes strongly suggests that thymic blast cells do not become pyknotic. The rate of small thymocyte production and disappearance was studied by pulse and repeated ³H-TdR labelling techniques combined with dye exclusion studies and pyknotic counts. The data from the repeated labelling experiment were analysed by use of a model based on the assumption of first order kinetics of small viable, dead, and pyknotic thymocytes. The rate of cell production was estimated to 1–6 %/hr whereas the rates of cell loss due to disintegration, i.e. supravital stainability and nuclear pyknosis, were calculated to 0–02 %/hr and 0–0006 %/hr respectively. Cell loss due to disintegration was less than 2 % of the total loss of small thymocytes. It was concluded that pyknotic counts are a useless method of assessing the cell death in the population of thymic blast cells and small thymocytes. On the basis of a model for thymocyte proliferation, production and loss it is suggested that about 45 % of the small viable thymocytes re-enter the generative cell pool, whereas about 55% disappear by emigration.     

IMPLICATION OF INVOLVEMENT OF RAT CHROMOSOME #2 IN SPONTANEOUS TRANSFORMATION OF THE RAT-2 CELL LINE

Using anin vitromodel for cell transformation, the relationship between specific chromosomal aberration and phenotypic changes was studied at different passages of Rat-2 cell line. A marker chromosome resulting from a translocation [t(2;7)] was found to be associated with focus formation in soft agar. Conversely, the loss of this marker chromosome was found to be associated with phenotypic reversion. These results suggest an association of this marker chromosome with phenotypic transformation for the Rat-cell line.     

SUBEPIDERMAL AIR SPACES: BASIS FOR THE PHENOTYPIC EXPRESSION OF THE ARGENTEUM MUTANT OF PISUM

The Argenteum (Arg) mutant of pea is characterized by a gray-green, silvery cast of the leaves and stipules. Light and electron microscopy showed that the nature of the silvery cast was due to extensive intercellular air space between the epidermis and palisade parenchyma. Adaxial epidermal strips from mutant leaves were free of palisade cell wall fragments, whereas similar strips from normal leaves had remnants of the torn palisade cells attached to the epidermal cell walls. The periphery of the epidermal-palisade cell wall interfaces showed a more electron-opaque middle lamella in the normal leaves than in the Arg mutant leaves.     

CHARACTERIZATION OF DEVELOPMENT IN MAIZE THROUGH THE USE OF MUTANTS. II. THE ABNORMAL GROWTH CONDITIONED BY THE KNOTTED MUTANT

The maize mutant Knotted (Kn) is characterized by hollow, finger-like outgrowths (knots) occurring mainly in the leaf blade. Portions of the ligule are displaced from the normal position to more distal locations within the blade. Knots apparently result from continued meristematic activity of isolated patches of cells surrounded by maturing tissue. Small knots appear to be centers of cell division. Epidermal cells overlying a small knot have been observed to undergo periclinal divisions. In addition to cell division, a reorientation of the axis of cell elongation is associated with knot formation. The pattern of knot distribution varies at different levels on the plant axis and within a leaf blade. From leaf 4 to leaf 10 or 11 the number of knots per leaf increases progressively, then declines in leaves initiated later. Knots always occur in association with lateral veins. The greatest number per vein occurs on the 3rd or 4th vein from the midrib. One plant developing from an X-rayed heterozygous seed possessed a sector of normal tissue bisecting the plant in a vertical plane passing through the midrib of each leaf except the top two. The normal sector was knot-free and had the ligule restored to the normal position. These observations suggest that cells with the characteristics of those from intercalary meristems occur throughout the blade in Knotted plants.     

Immunopotentiating Activity of Whole Cells and Mini-Cells of Salmonella typhimurium

Salmonella typhimurium strain 9 produces mini-cells during cell proliferation. Mini-cells are viable but cannot proliferate since they do not contain chromosomal DNA. Effects of whole cells and mini-cells of S. typhimurium on the immune responses were investigated, with the following results. Phagocytosis of peritoneal macrophages was enhanced by in vivo stimulation of both whole cells and mini-cells. Cellular immunity against L1210 cells (mouse leukemia cells) and Sarcoma 180 cells was also enhanced by both whole cells and mini-cells. Mini-cells slightly stimulated in vitro blast cell transformation of normal mouse lymphocytes. Whole cells of S. typhimurium induced antibody-forming cells to produce IgG of higher affinity but mini-cells did not. Mini-cells were not directly cytotoxic for normal lymphocytes or L1210 cells.     

Endocytosis and exocytosis of phytohemagglutinin (PHA) cell surface receptors of human lymphocytes during blast transformation

The ultrastructural distribution of T lymphocyte surface membrane receptors for phytohemag-glutinin (PHA) during blast transformation is examined using PHA covalently coupled to ferritin (PHA-Fe). Human peripheral blood lymphocytes from normal donors were enriched for T cells by nylon wool elution and cultured in vitro with PHA-Fe at a concentration known to cause maximal stimulation of DNA synthesis as measured by [³H]thymidine incorporation. Over the course of a 72 h incubation period, cell samples were harvested at regular intervals and examined by transmission electron microscopy. Within several minutes of culture at 37 °C the majority of the ferritin (Fe)-labeled PHA surface receptors on almost all cells undergo rapid endocytosis; some Fe label remains at the cell surface. After several hours, endocytotic vesicles containing Fe-labeled receptors coalesce and undergo condensation. Within 36–48 h, most endocytotic vesicles transform into multivesicular bodies (MVBs). After 48–72 h, 70–80% of the cells had the ultrastructural appearance of blast transformation as characterized by increased size, euchromatic nuclei, nucleolonema and polyribosomes. In 40 % of the blast cells the Fe-labeled MVBs are exocytosed to the cell surface; cytoplasmic MVBs in the remaining portion of the blasts and non-blast lymphocytes do not appear to undergo exocytosis. Although endocytosis and exocytosis of lymphocyte surface receptors during mitogen-induced blast transformation are observed, the role and significance of receptor redistribution to cell activation remains unclear.     

THE EVOLUTION OF PARASITISM IN RED ALGAE: CELLULAR INTERACTIONS OF ADELPHOPARASITES AND THEIR HOSTS1

In the initial stages of cell–cell interactions (spore germination and host penetration), the adelphoparasites Gardneriella tuberifera Kyl. and Gracilariophila oryzoides Setch. & Wilson form infection rhizoids that fuse directly with underlying host epidermal or cortical cells. In so doing, parasite nuclei and other organelles enter the cytoplasm of the host. The resulting heterokaryon may fuse with adjacent host cells either directly, via secondary pit connections, or by the dissolution or dislodgment of pit plugs from existing pit connections. The cell fusion events result in a heterokaryotic syncytium in which parasite nuclei replicate. In Gardneriella, formation of the syncytium induces surrounding host tissues to divide to form a photosynthetic callus. The internalized syncytium forms conjunctor and rhizoidal cells that fuse with host callus, eventually transforming the host callus into cells containing parasite nuclei. Gracilariophila does not induce surrounding host tissue to divide. Rather, division of the initial heterokaryotic tissue gives rise to the colorless mantle that protrudes from the host and forms reproductive structures. The heterokaryotic tissue also fuses with underlying host cells, thereby spreading parasite nuclei throughout adjacent host cells. In both these adelphoparasites, transformation of host cells by parasite nuclear invasion results in plastid dedifferentiation, an increase in mitochondria, autolysis of organelles, and accumulation of large amounts of floridean starch. The development and physiology of these parasites is similar to normal post-fertilization processes in the hosts that give rise to carposporophytes and suggests that these adelphoparasites may have originated from perturbations of developmental pathways involved in their host's post-fertilization development.     

THE EFFECT OF POLYCYTHAEMIC SERUM ON THE PROLIFERATION OF RAT BONE MARROW CELLS IN VITRO

The effect of experimental polycythaemia on the rate of proliferation of erythrocytic precursor cells was investigated by means of an in vitro technique. The serum obtained from polycythaemic rats was found to inhibit significantly ³H-thymidine incorporation in normal rat bone marrow cells in vitro, as compared with normal serum. Autoradiographic analysis revealed that this inhibition resulted from a reduction in the number of labelled bone marrow cells. The inhibition proved to be specific to the erythrocyte precursor cells; the labelling index was reduced in the erythrocytic cell population by 21–50% (P < 0.001) at different incubation times, while the effect on the granulocytic cell population was negligible. It is deduced that an inhibitor substance responsible for the effects observed is present in polycythaemic serum. It is proposed that this factor is the ‘erythrocytic chalone'. The results support the general view that triggering of stem cells is not the only mode of regulation of erythropoiesis, but that the rate of proliferation of the precursor cells in the erythron is also regulated.     

ISOLATION AND CHARACTERIZATION OF A CELL WALL‐DEFECTIVE MUTANT OF CHLAMYDOMONAS MONOICA (CHLOROPHYTA)1

Cell wall–defective strains of Chlamydomonas have played an important role in the development of transformation protocols for introducing exogenous DNA (foreign genes or cloned Chlamydomonas genes) into C. reinhardtii. To promote the development of similar protocols for transformation of the distantly related homothallic species, C. monoica, we used UV mutagenesis to obtain a mutant strain with a defective cell wall. The mutant, cw‐1, was first identified on the basis of irregular colony shape and was subsequently shown to have reduced plating efficiency and increased sensitivity to lysis by a non‐ionic detergent as compared with wild‐type cells. Tetrad analysis of crosses involving the cw‐1 mutant confirmed 2:2 segregation of the cw:cw⁺ phenotypes, indicating that the wall defect resulted from mutation of a single nuclear gene. The phenotype showed incomplete penetrance and variable expressivity. Although some cells had apparently normal cell walls as viewed by TEM, many cells of the cw‐1 strain had broken cell walls and others were protoplasts completely devoid of a cell wall. Several cw‐1 isolates obtained from crosses involving the original mutant strain showed a marked enhancement of the mutant phenotype and may prove especially useful for future work involving somatic cell fusions or development of transformation protocols.     

A Model of Oscillatory Blood Cell Counts in Chronic Myelogenous Leukaemia

In certain blood diseases, oscillations are found in blood cell counts. Particularly, such oscillations are sometimes found in chronic myelogenous leukaemia, and then occur in all the derived blood cell types: red blood cells, white blood cells, and platelets. It has been suggested that such oscillations arise because of an instability in the pluri-potential stem cell population, associated with its regulatory control system. In this paper, we consider how such oscillations can arise in a model of competition between normal (S) and genetically altered abnormal (A) stem cells, as the latter population grows at the expense of the former. We use an analytic model of long period oscillations to describe regions of oscillatory behaviour in the S–A phase plane, and give parametric criteria to describe when such oscillations will occur. We also describe a mechanism which can explain dynamically how the transformation from chronic phase to acute phase and blast crisis can occur.     

ANALYSIS OF THE KINETICS OF GRANULOSA CELL POPULATIONS IN THE MOUSE OVARY

The kinetics of granulosa cell populations in two types of follicles in ovaries of 28-day-old Bagg mice are investigated. The analysis includes estimations of mean values and standard deviations of the transit times (T_G1, T_S, T_G2 and T_C), the doubling time T_D, and the proliferative fraction p. First the percentage of labelled mitosis curve (PLM-curve) and the continuous labelling curve (CL-curve) are estimated. Then a hypothesis concerning the cell kinetics of the granulosa cells in the two follicle types is set up. The normal distribution is chosen to simulate the probability density functions of the transit times. On the basis of the hypothesis mathematical expressions for the PLM- and CL-curves are worked out. By fitting the calculated PLM-curve to the experimental one it is possible to estimate mean values and standard deviations of T_G1, T_S>, and T_G2. As a test of the hypothesis the CL-curve is calculated by means of the estimated parameter values and compared to the experimental one. The calculated PLM- and CL-curves are found to be in good agreement with the experimental data as far as both follicle types are concerned. It is concluded that the method is a useful procedure. The choice of a normal distribution does not imply a significant limitation of the method in these investigations. Moreover it is concluded that the hypothesis is plausible. This means, e.g., that the proliferative fraction is unity in the two follicle types and that there is no cell loss from the cell systems.     

Lymphotoxin production and blast cell transformation by cord blood lymphocytes: dissociated lymphocyte function in newborn infants

The correlation between phytohemagglutinin-induced blast cell transformation and production of lymphotoxin was studied in the cord blood lymphocytes of 17 normal newborn infants. Twelve normal adults served as controls. Blast cell transformation by newborn lymphocytes was somewhat greater than that of adults. By contrast, lymphotoxin production by the newborns was only 40% of the value for the adult controls. When a lymphotoxin index (ratio of lymphotoxin production to transformation) was calculated, it was found that, for any degree of transformation, newborn lymphocytes produced only about one-fourth as much lymphotoxin as did adult lymphocytes. The combination of normal blastogenesis with diminished lymphokine production represents a dissociation of in vitro lymphocyte function. This phenomenon, which appears to be a stage in the maturation of cellular immune competence, may in part explain the undue susceptibility of newborn infants to certain mycobacterial, fungal, and viral infections.     

T-lymphocyte behaviour modification in vitro by cancer patient sera

Cell mediated immune response blocking factor(s) in the serum of colon adenocarcinoma patients inhibit blast transformation of lymphocytes from normal individuals. The blocking capacity of the sera has been shown to correlate with the infiltration of the tumor. This correlation suggests that these phenomena may be mediated by identical serum factors and through a common cell receptor present in the lymphocytes of normal individuals.     

Transformations of osteoclast phenotype in ia rats cured of congenital osteopetrosis

The reduced bone resorption characteristic of osteopetrosis is accompanied in the incisors-absent (ia) rat mutation by a significant increase in osteoclasts of inactive (mutant) phenotype. Restoration of bone resorption in ia rats by transfer of spleen cells from normal littermates is preceded by a transformation of osteoclasts from mutant to normal phenotype. In this investigation the proportions of osteoclasts of normal phenotype have been determined by light microscopy in untreated ia and normal rats and in ia rats treated with various cell populations from normal rats. Significant increases in numbers of osteoclasts of normal phenotype were seen in the mutant skeleton soon after cell treatments that eventually restored bone resorption and cured the disease. No changes in osteoclast phenotype were seen after cell transfers that did not cure the disease. These data establish transformation of osteoclast phenotype as an early event in the recovery from osteopetrosis and suggest that determination of osteoclast phenotype is a reliable predictor of the success of normal cell populations to restore bone resorption in this mutation.     

Cytochemistry of gamma-glutamyltransferase in haemic cells and malignancies

Summary An improved cytochemical method for the demonstration of -glutamyltransferase is described. The enzyme was demonstrated in almost all normal leucocytes and in platelets. There was markedly reduced activity in most lymphoproliferative disorders. In a single case of Hodgkin's disease, T-lymphocytes showed slight to moderate activity contrasting with the marked activity displayed by Reed—Sternberg cells. The plasma cells of multiple myeloma and plasma cell leukaemia showed activity equal to or stronger than that of their normal counterparts. In acute myeloid leukaemia the positivity varied widely in blast cells, but was consistently increased in monoblasts of acute monoblastic leukaemia. -Glutamyltransferase may serve as a differentiation marker in the study of granulocytic and lymphocytic cell lineages.     

RADIOAUTOGRAPHIC STUDIES OF THE STEM CELL IN THE THYMUS OF THE IRRADIATED RAT

Radioautographic evidence is presented which characterizes the marrow derived stem cell which promotes thymic recovery following irradiation in the rat. These immigrant cells are similar in morphology to blood monocytes and have been called monocytoid, meaning monocyte-like in appearance. The typical cell had abundant pale staining cytoplasm and a nucleus with many invaginations and folds and a fine chromatin structure. There was no prominent nucleolus. The majority of these cells entered the thymus of the irradiated rat via the blood vessels into the septa and made their way through the connective tissue to the outer cortex. Three distinct morphological cell types appeared to be derived from the immigrant cells. These were fibrocyte-like cells which were located within the septa, macrophages located mainly within the medulla and septa, and large blast cells within the cortex, which proliferated giving rise to large thymocytes. The blast cells were characterized as having abundant moderately basophilic (and pyroninophilic) cytoplasm with a distinct cytoplasmic boundary, a large nucleus which still had invaginations and folds, a loose chromatin structure and one or more very prominent nucleoli. They were located in groups primarily within the outer cortex and often adjacent to blood vessels. They were found to be highly susceptible to damage in smear preparations. In contrast, their progeny, the large thymocytes were not highly susceptible to damage in smear preparations but teased out as large round cells with a highly basophilic rim of cytoplasm. The large thymocytes were precursors to medium and small cells. A radioautographic technique for 1 μ tissue sections is also described.     

Development of transgenic finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.) resistant to leaf blast disease

Finger millet plants conferring resistance to leaf blast disease have been developed by inserting a rice chitinase (chi11) gene through Agrobacterium-mediated transformation. Plasmid pHyg-Chi.11 harbouring the rice chitinase gene under the control of maize ubiquitin promoter was introduced into finger millet using Agrobacterium strain LBA4404 (pSB1). Transformed plants were selected and regenerated on hygromycin-supplemented medium. Transient expression of transgene was confirmed by GUS histochemical staining. The incorporation of rice chitinase gene in R₀ and R₁ progenies was confirmed by PCR and Southern blot analyses. Expression of chitinase gene in finger millet was confirmed by Western blot analysis with a barley chitinase antibody. A leaf blast assay was also performed by challenging the transgenic plants with spores of Pyricularia grisea. The frequency of transient expression was 16.3% to 19.3%. Stable frequency was 3.5% to 3.9%. Southern blot analysis confirmed the integration of 3.1 kb chitinase gene. Western blot analysis detected the presence of 35 kDa chitinase enzyme. Chitinase activity ranged from 19.4 to 24.8. In segregation analysis, the transgenic R₁ lines produced three resistant and one sensitive for hygromycin, confirming the normal Mendelian pattern of transgene segregation. Transgenic plants showed high level of resistance to leaf blast disease compared to control plants. This is the first study reporting the introduction of rice chitinase gene into finger millet for leaf blast resistance.     

THE 'TRANSITION PROBABILITY MODEL' AND THE REGULATION OF PROLIFERATION OF HUMAN DIPLOID CELL CULTURES DURING AGING

Genealogies derived from time-lapse cinemicrophotographic studies of aging human diploid cell cultures were analysed in terms of the ‘transition probability’ model. It was found that the distribution of intermitotic times obtained from middle passage cells deviated only slightly from that predicted by the model. In contrast, the plot for late passage cultures did not fit the predicted pattern and appeared to be composed of multiple curves. These changes are discussed in reference to cellular senescence as expressed by normal human diploid cells in vitro.     

QUANTIFICATION OF THE EFFECTS OF SPORELING COALESCENCE ON THE EARLY DEVELOPMENT OF GRACILARIA CHILENSIS (RHODOPHYTA)1

Sporeling coalescence in species of Gracilariales and Gigartinales is predicted to result in larger basal areas of growing disks as well as earlier initiation, increased abundance, and faster growth rates of erect shoots as compared to noncoalescent sporelings. These responses have been interpreted as providing mutual benefits for organisms living in aggregation, counterbalancing disadvantages associated with crowding. Quantitative evaluations of sporelings of Gracilaria chilensis failed to support several of these predictions. Sporelings were grown in the laboratory from a range of single sporelings to coalescent masses of 20 sporelings. Coalescent sporeling masses of G. chilensis exhibited larger basal areas than noncoalescent ones, but because the specific growth rates were inversely related to the original number of carpospores, no significant differences in actual area increments, during most of the experiment, were found among sporelings derived from one, two, or three to five coalescing sporelings. Initiation of erect shoots occurred at a similar time, regardless of their origin, i.e. coalescent or noncoalescent. Abundance of erect shoots was only loosely related to the number of coalescing sporelings. Even though by the end of the experiment (week 18), the total length of the longer erect shoots arising from coalescent sporeling masses was significantly greater than that of shoots arising from noncoalescent sporelings, total length was independent of the original number of coalescing sporelings. Furthermore, specific elongation rates between week 12 and week 18 were significantly greater for noncoalescent sporelings than for coalescent sporeling masses. Quantitative screening of other species seems necessary before generalizations on the ecological advantages of sporeling coalescence in seaweeds can be made.