Newly-formed Cells Research Articles

Antibody against phosphorylated proteins (MPM-2) recognizes mitotic microtubules in endosperm cells of higher plant Haemanthus.

In diverse cell types, monoclonal antibody MPM-2 recognizes a class of phosphorylated proteins related to microtubule organizing centers and abundant during mitosis. We have used this antibody in an attempt to identify the spatial and temporal localization of putative microtubule organizing centers in endosperm cells of the higher plant Haemanthus. Our results show that MPM-2 recognized epitope is present in interphase cells and enriched in mitotic cells. In interphase the antibody usually stains cytoplasmic granules. During the interphase-prophase transition immunoreactive material appears in the nucleus, at the nuclear envelope, and in association with microtubules. Concomitantly, we observed an increase of immunoreactivity of the cytoplasm. During mitosis the phosphorproteins recognized by MPM-2 are detected in the cytoplasm, in association with microtubules of the spindle, the phragmoplast, and in the newly-formed cell plate. After completion of mitosis, only the cell plate and cytoplasmic granules are MPM-2 positive. Extraction of the cells with Triton X-100 prior to fixation removes staining of the cytoplasm by MPM-2. The detergent resistant immunoreactive material remains associated with surrounding the nucleus microtubules of the prophase spindle, the core of kinetochore fibers, and the phragmoplast. In the phragmoplast, however, segments of microtubules which are distal to the cell plate are depleted of MPM-2. These data demonstrate that microtubule arrays of endosperm cells are phosphorylated during mitosis. Thus, similar to animal cells, interphase and mitotic microtubules of higher plants have different properties. Additionally, the localization of detergent resistant MPM-2 antigen points to the difference in microtubule nucleation/organization between higher plant and animal cells.

Growth-rate-independent killing by ciprofloxacin of biofilm-derived Staphylococcus epidermidis; evidence for cell-cycle dependency.

Cell culture methods that allow culture of Staphylococcus epidermidis biofilms at controlled growth rates were used to examine susceptibility to ciprofloxacin. Changes in biofilm susceptibility, dependent upon growth rate, were compared with those for suspended populations grown in chemostat, and also for newly-formed daughter cells shed from the biofilm during its growth and development. Susceptibility increased for intact and resuspended biofilms, and also for planktonic cultures, with increases in growth rate. The dependence of susceptibility upon growth rate was greatest for slow growing cells (mu, 0.01-0.15/h). At any particular growth rate, biofilms appeared more susceptible than their planktonic counterparts. Newly-formed daughter cells were relatively tolerant to ciprofloxacin at all rates of growth. Lack of growth rate dependency for the newly-formed cells suggested a role for the cell-division cycle in determining resistance. This was confirmed by examining the susceptibility of S. epidermidis throughout batch cultures with cell division synchronized. Perfusion of various steady-state biofilms with ciprofloxacin demonstrated killing of the adherent population even at much reduced rates of growth.

Capture of Rotifers byAcaulopage Pectospora, and Further Evidence of its Similarity toZoophagus Insidians

Acaulopage pectospora is a nematode-trapping zoopagaceous fungus in which the hyphae bear a number of short lateral branches (ca 10-25 tm long, 2.5-3.0 Am wide) as trapping organs. Each branch terminates in a small, globose knob from which an adhesive is squeezed out to capture nematodes (Drechsler, 1962). This species is unique in the genus Acaulopage in that all other known species capture amoebae using the entire surface of their undifferentiated hyphae. The extremely large elongated conidia (ca 180-240 x 7-14 ,um) are also exceptional for the genus. Recently, Saikawa and Morikawa (1985) reported several ultrastructural aspects of the fungus as follows: (1) the trap's cytoplasm was occupied by large electron-dense vesicles (0.4-0.8 ,um) before secretion of the adhesive; (2) the secreted adhesive derived from the large vesicles had a high electron density and contained a number of bubbles; (3) the cell walls of the traps became doubled after the secretion of the adhesive and an aliquot of the adhesive remained in the intervening spaces between the outer and newly-formed inner cell walls; (4) the outer wall of the terminal knob exhibited a rimose appearance. These ultrastructural characteristics in A. pectospora are similar to those of Z. insidians (Whisler and Travland, 1974) except that the rimose appearance in the terminal knob is replaced in the latter by the fine ridges in the apical, rounded end of the short branches. These similarities led Saikawa and Morikawa (1985) previously to suggest a close relationship between the two species. In the present study, A. pectospora was tested for its ability to capture rotifers instead of nematodes in an aquatic environment, and Z. insidians was tested for its ability to trap nematodes. MATERIALS AND METHODS

SURFACE ULTRASTRUCTURE OF CULTURES OF AFFECTED PSORIATIC KERATINOCYTES, UNAFFECTED PSORIATIC KERATINOCYTES AND NORMAL KERATINOCYTES

The surface structure of 11-12 days old cultures from biopsies of normal skin and uninvolved psoriatic skin was investigated by scanning electron microscopy. The keratinocytes formed flat, thin, and well-organized layers of elongated, tightly apposed cells. Newly-formed cells with few and short microplicae and microvilli were seen at the colony periphery. On the maturing cells towards the colony center, the number and size of microplicae or microvilli increased gradually. No differences were found between the structures on normal keratinocytes and keratinocytes from uninvolved psoriatic skin, but in keratinocytes from involved psoriatic skin glomerulus-like rolled microplicae occasionally were found.

Erythropoietin assay: present status of methods, pitfalls, and results in polycythemic disorders.

Mammalian erythropoiesis is regulated primarily by the hormone erythropoietin (ESP). Studies of ESF have provided information about its biochemistry and its role in regulating hemoglobin synthesis. Such studies rely on assays for erythropoietic activity in biological fluid. The assay which has proven most valuable and is used most widely is based upon the incorporation of radioactive iron into newly-formed red cells of polycythemic mice. While this assay has gained wide acceptance, it is expensive, cumbersome, imprecise, and insensitive, capable of reliably detecting no less than 50 milliunits of erythropoietin. Improvements in assay techniques will require new methodology relying primarily on immunologic recognition for the determination of hormone activity. Currently under development and in experimental use are radioimmunoassays and a hemagglutination inhibition assay. While work has progressed in these areas, these assays are not of proven value at present and meaningful physiological correlations have not emerged from their use. Alternatively, assays for hormone activity using suspensions of hematopoietic cells and the measurement of incorporation of radioactive isotopes into hemoglobin have provided both improvement in sensitivity and precision. The disadvantage of these types of assays is that they are sensitive to factors other than ESF and may give misleading information, depending on whether the factors present stimulate or inhibit cellular proliferation and hemoglobin synthesis. While such techniques may provide a temporary solution to some problems associated with assaying ESF for purification or physiological studies, they are not the best answer to the overall problem of hormone detection and characterization. The most important contribution to this field will be the availability of large amounts of highly purified and well-characterized ESF.

Tritiated thymidine autoradiographic study on cellular migration in the gastric gland of the golden hamster.

Ten hamsters received repeated injections of 3H-thymidine for 4 days and were allowed to survive for 7, 28, 42 and 100 days. Changes in spatial distribution of the labelled cells and in labelling indices of each cell line in the gastric glands were studied at various days after 3H-thymidine, the labelled parietal cells and the mucous neck cells were concentrated at the neck area. Starting from the neck area, they migrated an average of 3 micra downwards per day. By 42 days, they reached the middle level of the glands, where the labelled mucous neck cells decreased but the labelled chief cells increased in number. The differentiation of the chief cell then appears to take place at the middle level of the glands through transformation of the migratory mucous neck cells. After 4 days of the labelling, about 1.8% of the chief cells located in the lower part of the glands was found to undergo in situ replication. This indicates that the renewal of this cell type is partly assured by its own mitotic activity. The foveolar cell--the future surface epithelium--seems to migrate upwards along the long axis of the glandular tubule in the pipe line system, which means "first produced, first migrates". After migrating out from the neck area, the parietal cell and the mucous neck cell (the future chief cell) take an average of 200 days to reach the lower end of the glands. In the process of migration, however, the cells produced contemporaneously at the neck area became scatteringly spread from the neck towards the bottom of the gland. The time required for the newly-formed cells to reach the lower end of the gland varied between 100 and 300 days. In the gastric glands the cells first produced at the neck area do not first reach the lower end of the glands. This mode of random migration is referred to as the "stochastic flow system". As one of the probable factors which disturb the pipe line flow of downward cell migration, cellular movements perpendicular to the long axis of the glandular tubule were suggested to occur at random at any level of the gastric glands.

Alternative mechanisms for homeostatic regulation of mammalian cell populations

A model for homeostatic regulation in mammalian tissues is analyzed. The model treats resting and active dividing cells, immature and mature non-dividing cells as separate populations. In the model, regulation is accomplished through control of the proportion of newly-formed cells that will become non-dividers. Four possible regulating substances, produced by dividing cells, non-dividing cells, mature non-dividing cells, and newly-formed cells respectively, are considered. Stability theorems are provided. System behavior in each instance depends on the relative values of the rate at which cells divide and the rate at which non-dividers die.

Pairwise loss of mitotic ability by human diploid fibroblasts

Non-transformed human diploid fibroblasts, like WI-38, cannot be maintained as permanent cell lines. With successive divisions in culture, the cells lose the ability to synthesize DNA. This loss may be due to inheritance, to a shared environment, or to interaction among offspring. We studied the pattern of DNA synthesis in WI-38 daughter pairs. A synchronized population in early prophase was seeded in cloning medium containing tritiated thymidine. Radioautographs were made after three days of incubation. We found that the loss of ability to undergo DNA synthesis was correlated between the members of daughter pairs. Daughter pairs in which one but not both of the cells had incorporated the labelled thymidine were significantly less frequent than if the loss of ability to synthesize DNA were to occur at random. The spatial pattern of loss is consistent with the release of a diffusible inhibitor of DNA synthesis by each newly-formed cell. Daughter pairs in which one or both of the cells incorporate thymidine occur with increasing frequency as the inter-nuclear distance between pair members increases. Interaction between daughter cells is responsible in part for the finite lifespan of human diploid fibroblasts.

Biochemical changes during the development of cork spot of apples

Biochemical changes occurring during the development of cork spot of ‘York Imperial’ were investigated. At the first visible sign of the disorder, the rate of ethylene production increases in the affected tissue. Respiration also increases, acetate being the major respiratory substrate rather than glucose. Protein synthesis, pectin synthesis, and the movement of inorganic ions into the tissue follow. During the time the chemical changes are taking place in the tissue, abnormal cell division is initiated, packing the newly-formed cells into the intercellular spaces. At the final stage of development, the tissue becomes brown and appears as a firm brown spot in the flesh of the apple. Cork spot is somewhat different from bitter pit in that the spots appear early in the season, the affected tissue is deeper in the flesh, and firmer. The chemical changes discovered so far in both disorders, however, appear to be similar. We consider the abnormal chemical changes that occur in both disorders to be common to diseases and injuries and not specific for either cork spot or bitter pit.

The intracellular distribution of mass during ontogeny of pollen in Tradescantia L

1. 1. Changes in the distribution of intracellular mass during ontogeny of pollen grains, as recorded by the application of Engström's X-ray absorption technique, are reported for the first time. 2. 2. During development of the mature pollen grain the greatest relative mass is associated with the chromosomes early in the process (pachytene through metaphase I). 3. 3. The extranuclear portions of the cells attain absorption (mass) characteristics similar to those of early prophase I chromosomes beginning approximately at late anaphase I—early telophase I. 4. 4. Cytokinesis, at telophase I and II, is strikingly associated with sharply localized heavy X-ray absorption in the newly-formed cell walls. 5. 5. By the end of telophase II, each microsporocyte has produced four microspores whose distinctive vacuome is recorded by minimal X-ray absorption. 6. 6. In the microspore, and particularly in the vegetative (tube) nucleus of the early binucleate pollen grain, the nucleolus becomes distinctly differentiated from the remainder of the nuclear structures by its comparatively intense X-ray absorption. 7. 7. During the later phases of ontogeny of the pollen grain, there appears to be a gradual increase in mass (X-ray absorption) in the cytoplasm. 8. 8. In the mature binucleate pollen grain high X-ray absorption is associated with extranuclear structure (cytoplasm and cell wall). Minimal absorption is recorded at this stage in both of the nuclei. 9. 9. Specific difficulties encountered in investigating microsporocyte (pollenmother-cell) material with this X-ray technique are briefly reviewed.

On the Reactions of the Stellate Cells of the Rat Liver to Trypan Blue

1. Albino rats, weighing 150-200gm (3-5 months of age), were treated with repeated subcutaneous injections of 1cc. of 1% aqueous solution of trypan blue (E. MERK), and the reactions of reticulo-endothelial cells in the liver, spleen, lymph nodes, bone marrow and other organs have been observed.2. When injection of trypan blue had been repeated twice or once weekly, or fortnightly, foci of round cells began to appear in the interlobular spaces of the liver after 2-3 months and grew rapidly in size thereafter, so that after 7-8 months they became visible by the naked eye (Figs. 1-2). The majority of the cells constituting the newly-formed cell foci were storing trypan blue in relatively large granules as did the stellate cells of KUPFFER (Fig. 3). Splenectomy, that had been performed one month prior to the injection of trypan blue, enhanced the formation of foci of round cells in the liver to some extent (Fig. 1).3. After treatment with twice daily injections of trypan blue for 7-14 days, the stellate cells of the liver became markedly swollen, assuming an oval or rounded shape, and were in active proliferation. Mitotic figures were frequently seen in the swollen stellate cells (Figs. 5-6). In organs other than the liver, however, such proliferative reaction of reticuloendothelial cells occurred only to a much lesser degree (Fig. 7).4. The swollen stellate cells of the liver, which had became desquamated from the sinusoidal wall, appeared to be transferred by the blood stream to the lung where large round cells stroring trypan blue greatly increased in number (Figs. 9-10). However, some of these cells immediately migrated into the interlobular spaces (Fig. 4).5. The stellate cells of the liver in mitosis usually did not contain trypan blue granules in their cell body, but some of the dividing cells apparently showed dye-storing ability (Fig. 6). In the former case, the stellate cells in mitosis were discriminated from other cells by the form and size of their cell body and by the site of their occurrence (Fig. 5).6. The claim of previous investigators such as SSYSSOJEW (1926) and MALYSCHEW (1927), that stellate cells of Kupffer and other reticuloendothelial cells may transform into blood cells during the processes of their proliferation, could not be verified in the present observations.7. On the basis of the findings outlined above, it is evident that active proliferation of stellate cells of the liver may be induced by repeated injections of trypan blue, and it seem reasonable to consider proliferation of stellate cells to be one of the major factors responsible for the formation of foci of round cells in the interlobular spaces of the liver.

MORPHOLOGICAL DEVELOPMENT OF NODULES ON CARAGANA ARBORESCENS LAM.

Developmental stages of nodules on Caragana arborescens, a woody perennial leguminous species, were studied. Root hair invasion and early cortical infection conformed to the well-known description of herbaceous leguminous nodules having apical meristems, except that a relatively smaller number of cells was infected. In the region of maturation behind the nodule meristem the infection threads remained active throughout the growth of the nodule and served to spread the infection to newly-formed cells. In mature nodules the distribution of red pigment, leghemoglobin, corresponded exactly with the location of the active bacteroidal tissue in the apical zones. Endodermal thickening of the vascular sheath and suberization of the nodule endodermis apparently restricted nutrient diffusion and aeration within the proximal areas. Senescence commenced relatively early, presumably as a result of these factors. However, the thickening of cell walls in these areas delayed, or prevented, necrosis; and a protective periderm which supplanted the outer cortex enhanced longevity of the nodule. Nodule rootlets projecting from six- to eight-month-old nodules were observed. The development of these rootlets from lateral branches of vascular strands and from terminal extensions of the apical tips of vascular bundles within the nodule is described.

The Behaviour of the Golgi bodies during nuclear division, with special reference to Amitosis in Dytiscus marginalis.

ABSTRACT Dictyokinesis, or division of the Golgi bodies during cell division, has recently been worked out by Professor J. Bronte Gatenby and the present writer in the male germ-cells of several animals (5). It was found that the distribution of the Golgi elements, or dictyosomes, to both halves of a dividing cell was a very haphazard process and was unaccompanied by any splitting of the dictyosomes such as occurs in the case of the chromosomes. In the cricket Stenobothrus, the dictyosomes become scattered in the cytoplasm of the spermatogonium before cell division takes place, and they remain in this condition during meiosis, so that approximately a half become contained within each of the newly-formed cells. A different process, however, occurs in the Mammals, Mus and Cavia, and in the Molluscs, Helix and Limnaea. The Golgi bodies in the spermatogonia of these types consist of a number of dictyosomes arranged around the archoplasm, inside which is the centrosome. As this latter organ divides, preparatory to the formation of the spindle, its two constituent parts separate and carry with them to both ends of the cell, approximately half of the archoplasm, still with the dictyosomes attached. During late prophase, the dictyosomes become temporarily detached from the archoplasm and scattered throughout the cell, and then at the late telophase they collect together again around the archoplasm.

Biological Notes

OOSPORES OF “VoLVOX MINOR.”—Dr. Kirchner, in the recent part of Cohn's “Beiträge zur Biologic der Pflanzen,” describes the germination of the oospores, and in this supplements the important contribution made by Cohn himself to our knowledge of this interesting plant in the first volume of the same work. The first appearance of germination was in February. The contents of the oospores during a rapid swelling out of the endospore, made their appearance through the ruptured exospore, and soon presented a sphere-shaped body, which then divided into two portions, these being perpendicular to one another. The newly-formed cells so separate from one another that they hang together by their ends. These ends form the one pole of the later-to-be-developed ball-like colony; the other pole is afterwards closed in, when the maximum of the cells is attained. Each oospore thus gives rise through cell division, followed by the characteristic cell displacement, to a new volvox colony. The fact of V. minor being diœcious was given as a character to distinguish it from V. globator, but this, according to the author, does not hold true; both colonies seem to pass through a purely female stage and afterwards through a male stage, each colony being bi-sexual.