HarpinPss elicited early defense responses of tobacco cells and involvement of calcium]

HarpinPss can induce hypersensitive reaction (HR) in tobacco leaves. As superoxide dismutase can inhibit but catalase can not inhibit the development of HR induced by harpinPss, superoxide anion is required for this response. HarpinPss can also induce the release of active oxygen and extracellular alkalinization, two early defence responses in tobacco suspension culture. Diphenylene iodoium, can completely inhibit the induction of HR in tobacco leaves, and the release of active oxygen in the suspension culture system, superoxide anion in these system may be produced by the activation of NADPH oxidase. Ethyleneglycol-bis (beta-aminoethyl) N, N, N'N'-tetraacetic acid (EGTA) can inhibit the development of harpinPss-induced HR and these two early defence responses in suspension culture system. Adding Ca2+ into the medium again, these responses can return to normal level in a short time. Lanthanum chloride, verapamil, neomycin, U-73122, and LiCl can also inhibit these harpinPss-induced responses. Therefore, the influx of Ca2+ mediated by calcium channel and the release of Ca2+ from internal Ca2+ pool may be involved in the two early defense responses induced by harpinPss. Cycloheximide and actinomycin D have no effect on the release of active oxygen but can inhibit harpinPss-induced HR even added them in the intermediate process for inducing HR. It indicates superoxide is just a trigger for HR, and HR is a more complex process that needs the sustained expression of some genes.     

Cytochemistry of dipeptidylaminopeptidase IV and II in normal and neoplastic lymphoid cells

Cytochemical methods were used to determine the distribution of dipeptidylaminopeptidase IV (DAP IV) and II (DAP II) in lymphoid cell populations from patients with lymphoproliferative diseases. Special attention was paid to unusual intracellular distribution patterns which might correlate with the presence of various membrane markers. In healthy patients, about 50% of the circulating lymphocytes were found to be positive to both reactions, the intracellular distribution patterns being variable. The DAP IV reaction was negative in all B-CLL cases. In 2 cases of T-CLL with phenotype E+ OKT3+T4-T8+ one was negative and one was weakly positive, while two cases of T-CLL with phenotype E+ OKT3+T4+T8- were both strongly positive. The other non-T lymphoproliferative diseases studied were negative for DAP IV, while one T-ALL and three T-lymphoma cases showed a strong granular or diffuse distribution. The DAP II reaction was strongly positive in all the T lymphoproliferative diseases studied, irrespective of their immunological phenotype. This reaction was also weakly positive in some cases of plasmocytoma and lymphoplasmacytoid lymphoma.     

Transferrin receptor cycling by human lymphoid cells: lack of effect from inhibition of microtubule assembly

Flow cytometry was used to follow the uptake and release of fluorescein-conjugated ferro-transferrin by CCRF-CEM human T - leukaemia cells on an individual cell basis. Pretreatment with the stathmokinetic agent vincristine for 5 hr had no effects on the cycling of transferrin, although microscopic examination of the cells showed a substantial proportion to be arrested in metaphase with cytoplasmic dispersal of tubulin, indicating inhibition of microtubule assembly. Tubulin is therefore unlikely to play a major role in the transferrin cycle of exponentially-growing cells, despite earlier reports that the cytoskeleton is involved in receptor clustering and down regulation.     

Granulocytic sarcoma with pleural involvement. Identification of neoplastic cells with cytochemistry

Malignant cells were detected in the pleural effusion of a patient with three separate primary malignancies. These cells were judged by conventional morphologic studies to be poorly differentiated cells, but cytochemical studies showed them to be granulocytic precursor cells. The use of cytochemical or immunochemical techniques may be most practical for the cytodiagnosis of malignant cells in serous effusions.     

Calcium movements and intracellular calcium distribution in neoplastic GH3 cells

Experiments were carried out to investigate the nature of the calcium homeostatic mechanisms in neoplastic GH3 rat pituitary cells. GH3 cells grown and maintained in Ham's F10 culture medium contained 35 nmoles calcium/mg cell protein. When stimulated by thyrotropin releasing hormone (TRH) or elevated K+ concentrations, only the latter caused cell calcium levels to rise although both resulted in hormone release. When exposed to EGTA, the GH3 cells lost calcium. When the temperature was lowered to 4 degrees C, the cells gained calcium and when rewarmed were able to extrude the previously accumulated calcium. The increased cell calcium following cold exposure could be blocked by prior treatment with rotenone. If rotenone was added subsequent to the cold exposure, it did not block the extrusion seen upon rewarming. In the absence of glucose in the medium, the GH3 cells took up more calcium upon exposure to 4 degrees C, and upon rewarming the cells could not return to their previous low levels. There are thus significant differences in calcium homeostasis between the neoplastic GH3 cells and their normal pituitary counterparts. When intracellular calcium was localized with the potassium pyroantimonate technique, there was calcium found in/on mitochondria, membrane bound vesicles and plasma membrane. Nuclear staining was sparse, and nucleolar staining was virtually absent. Upon stimulation with TRH, there was a decrease in mitochondrial calcium along with increases in both plasma membrane and nucleolar calcium levels. Since total calcium is unchanged, this indicates a significant calcium redistribution in response to TRH. The increased nucleolar calcium may reflect a calcium dependent increase in mRNA synthesis as has been reported. Since TRH presumably acts at a surface receptor, the increased plasma membrane calcium might be functionally related to receptor activation.     

Natural cytotoxicity in rats: Radiation-induced changes in the early killing of allogeneic cells

In some strain combinations among inbred tats intravenously injected ⁵¹Cr-labeled lymphocytes are rapidly destroyed in substantial numbers by unsensitized allogeneic hosts. This phenomenon has been referred to as natural cytotoxicity (NC) and is characterized by decreased lymph node radioactivity, increased kidney and urine radioactivity, and to a lesser extent increased liver radioactivity in allogeneic hosts, when compared with the distribution of label in syngeneic recipients of the same cell suspension. A single exposure to 800 rad either 1 or 7 days before the injection of ⁵¹Cr-labeled lymphocytes effected a reduction in NC as defined by all the above parameters in a strain combination exhibiting high NC. The same dosage of radiation abolished NC in a strain combination exhibiting intermediate → low NC. Because NC was not always completely abolished, the phenomenon was held to be partially radiosensitive. An increased accumulation of ⁵¹Cr-labeled lymphocytes was seen in the lymph nodes of both syngeneic and allogeneic irradiated hosts when compared with nonirradiated controls, although the increase was greater in allogeneic than in syngeneic hosts. This increased colonization in the lymph nodes of irradiated hosts seemed unlikely to be due to an increase in the available “space” in the lymph nodes following irradiation.     

Augmentation of zinc ion stimulation of lymphoid cells by calcium and lithium

Stimulation of hamster lymph node cells by optimal concentrations of ZnCl₂ (10 μM) was found to be enhanced by addition of 1–25 mM LiCl to the serum-free cultures. Maximal enhancement occurred at 10 mM Li⁺. Similar concentrations of either KCl or NaCl did not potentiate stimulation. Addition of 1 mM CaCl₂, but not 1–25 mM MgCl₂, also potentiated Zn²⁺ stimulation of lymph node cells. When the cultures were supplemented with 1 mM Ca²⁺ + 10 mM Li⁺, a synergistic potentiation of Zn²⁺ stimulation occurred. In addition, the dose response curve for Zn²⁺ was shifted such that maximal stimulation occurred at 100–250 μM Zn²⁺, a concentration of Zn²⁺ which was toxic for the unsupplemented cultures. In Ca²⁺ + Li⁺ supplemented cultures, Zn²⁺ stimulated [³H]thymidine incorporation to levels comparable to those obtained when hamster lymphoid cells were stimulated with lectins. In addition to Zn²⁺ stimulation, Ca²⁺ + Li⁺ supplemented medium also enhanced Hg²⁺ stimulation of hamster lymph node cells but did not change the dose response curve for Hg²⁺. Therefore, the observed ionic effects on Zn²⁺ stimulation of lymphocytes were unique to this mitogen, when compared to either Hg²⁺ stimulation or previously reported lectin stimulation of hamster lymphoid cells.     

In vivo cytotoxic responses induced by allogeneic normal and neoplastic cells in mice: relative lack of immunogenicity of B16 melanoma cells

It has been previously reported from this laboratory that the C57BL/6 melanoma B16 will grow in allogeneic mice provided that donor-derived passenger leukocytes are first removed by in vitro passage of the tumor cells. In this paper we report that allogeneic and syngeneic mice support similar survival of [125I]iododeoxyuridine-labeled B16 cells (125I-B16 cells) as indicated by whole-body retention of 125I. B16 failed to induce the early cytotoxic response which was induced by semiallogeneic peritoneal cells (PC) and by two other allogeneic tumors (EL4 lymphoma and MC-2 sarcoma). They were nevertheless rapidly destroyed by the response induced by PC or EL4. B16 did show some immunogenicity as indicated by regression of some primary B16 tumors (45% ip and the majority of footpad tumors) and by the slow cytotoxic response to secondary 125I-B16 challenge following preimmunization with live or irradiated B16 cells. An equivalent response was induced by preimmunization with a 100-fold lower dose of PC. These findings are discussed in relation to the requirement for inductive stimuli, in addition to antigen, for the stimulation of in vivo cytotoxic responses.     

Role of extracellular calcium in the hyperthermic killing of CHL V79 cells

Two major questions are addressed by this study: Can an influx of calcium ion sensitize CHL V79 cells to hyperthermia, and, if so, does this occur during heating and does it play a crucial role in cell death? V79 cells are sensitized to hyperthermia by the calcium ionophore A23187 which also induces an influx of calcium at both 37 and 43 degrees C. Sensitization is at least partially dependent on the presence of extracellular calcium. In the absence of A23187, survival is independent of calcium concentration (from 0 to 25 mM) during heating, which differs from the behavior of hepatocytes which are sensitized to hyperthermia by 15 mM CaCl2. Calcium influx, as assayed by uptake of 45Ca measured after washing in LaCl3, is detectable in 3 mM CaCl2 only after 30 min at 45 degrees C, an exposure which reduces reproductive survival to less than 0.1%. Calcium uptake reaches 6 nmol/10(6) cells after 180 min at 45 degrees C. This is not due to a general loss of membrane permeability since there is no trypan blue staining during this time. In 15 mM CaCl2, influx occurs earlier (15 min) but still succeeds the loss of reproductive survival which is less than 1% at this time. Uptake is much higher in 15 mM CaCl2, reaching 10 nmol/10(6) cells by 30 min and 25 nmol/10(6) cells at 180 min, but the temporal pattern of uptake does not correlate with loss of reproductive survival. Thus, although A23187 sensitizes V79 cells to hyperthermia, probably by increased influx of calcium ion, and increased influx occurs during exposure to 45 degrees C, influx is not a crucial early event in the killing of V79 cells. This does not eliminate the possibility of intracellular calcium redistribution during hyperthermia.     

Synergism of v-myc and v-Ha-ras in the in vitro neoplastic progression of murine lymphoid cells.

Murine bone marrow was either singly or doubly infected with retroviral vectors expressing v-myc (OK10) or v-Ha-ras. The infected bone marrow was cultured in a system that supports the long-term growth of B-lineage lymphoid cells. While the v-myc vector by itself had no apparent effect on lymphoid culture establishment and growth, infection with the v-Ha-ras vector or coinfection with both v-myc and v-Ha-ras vectors led to the appearance of growth-stimulated cell populations. Clonal pre-B-cell lines stably expressing v-Ha-ras alone or both v-myc and v-Ha-ras grew out of these cultures. In comparison with cell lines expressing v-Ha-ras alone, cell lines expressing both v-myc and v-Ha-ras grew to higher densities, had reduced dependence on a feeder layer for growth, and had a marked increase in ability to grow in soft-agar medium. The cell lines expressing both oncogenes were highly tumorigenic in syngeneic animals. These experiments show that the v-myc oncogene in synergy with v-Ha-ras can play a direct role in the in vitro transformation of murine B lymphoid cells.     

Proteins synthesized in lymphoid cells early after activation by phytohemagglutinin

A double-isotope labeling approach has been employed in an attempt to identify the proteins synthesized by lymphocytes early after stimulation by phytohemagglutinin (PHA). The earliest effect of PHA, within the first hour, was the induction of large aggregates of cellular proteins, which were not dissociated by 1% sodium dodecyl sulfate (SDS) in the absence of β-mercaptoethanol. These aggregates were composed of proteins of molecular weight approximately 70,000, but they did not include PHA. The aggregates were made up of preexisting as well as newly synthesized cellular proteins. Subsequently, within the first 2 hr after the addition of PHA, there was a nonspecific stimulation of protein synthesis. This was followed by the preferential synthesis of several classes of proteins including at least one group of nuclear proteins. The structural changes described here are among the earliest events known to occur within the lymphoid cell after its interaction with PHA.     

Vitamin C decreases intracellular calcium level in human lymphoid cells.

Human lymphocytes have low levels of many antioxidant enzymes however they are know to concentrate vitamin C. Cell injury, including oxidative stress effects, is associated with calcium influx so the influence of vitamin C on the maintenance of calcium levels in leukocytes was studied. Incubation of Molt-3 human lymphoblastoid cells with physiologically relevant concentrations of vitamin C and the calcium ionophore A23187 reversed the calcium influx and increased nuclear protein level associated with the ionophore alone. It is concluded that intracellular vitamin C can inhibit calcium influx into leukocytes so helping to minimise cell damage.     

Thromboxane-induced pulmonary vasoconstriction: involvement of calcium

Infusion of tert-butyl hydroperoxide (t-bu-OOH) or arachidonic acid into rabbit pulmonary arteries stimulated thromboxane B2 (TxB2) production and caused pulmonary vasoconstriction. Both phenomena were blocked by cyclooxygenase inhibitors or a thromboxane synthase inhibitor. The increase in pulmonary arterial pressure caused by either t-bu-OOH or arachidonic acid infusion correlated with the concentration of TxB2 in the effluent perfusate. The concentration of TxB2 in the effluent perfusate, however, was always 10-fold greater after arachidonic acid infusion. In the rabbit pulmonary vascular bed lipoxygenase products did not appear involved in the vasoactive response to t-bu-OOH or exogenous arachidonic acid infusion. Calcium entry blockers or a calcium-free perfusate prevented the thromboxane-induced pulmonary vasoconstriction. Calmodulin inhibitors also blocked the pulmonary vasoconstriction induced by t-bu-OOH without affecting the production of TxB2 or prostacyclin. These results suggest that thromboxane causes pulmonary vasoconstriction by increasing cytosol calcium concentration.     

Iron-induced accumulation of hepatic metallothionein: the lack of glucocorticoid involvement

The process(es) by which parenteral iron effects the accumulation of hepatic metallothionein (MT) is not known. The present study examined glucocorticoids as potential mediators of this process. Chicks were given either one injection (ip) of iron (+1FE) at 10 mg Fe/kg, two injections of iron (+2FE) given 24 hr apart, or a single injection of saline. Plasma corticosterone was evaluated at various times following the last injection. Plasma corticosterone increased approximately 50% following +1FE but more than 200% at 2 and 4 hr following a second injection of iron (+2FE). Plasma zinc showed a transient increase followed by a considerable depression. Coincidentally, the accumulation (determined at 24 hr) of zinc MT in liver of +2FE chicks was three times higher than that of +1FE chicks. In another experiment, markedly greater changes, at similar time intervals, in plasma corticosterone were effected by multiple subcutaneous injections of adrenocorticotropic hormone (ACTH) (either 5 IU ACTH or 20 IU ACTH/kg). Subsequent analysis of hepatic zinc MT showed only minor changes as a result of ACTH injections. These results indicate that a change in the plasma glucocorticoid corticosterone is not a primary component in the process(es) by which parenteral iron effects an increase in hepatic zinc MT.     

Dexamethasone-induced inositol 1,4,5-trisphosphate receptor elevation in murine lymphoma cells is not required for dexamethasone-mediated calcium elevation and apoptosis

Glucocorticosteroid hormones, including dexamethasone, have diverse effects on immature lymphocyte function that ultimately lead to cell death. Previous studies established that glucocorticoid-induced alterations in intracellular calcium homeostasis promote apoptosis, but the mechanism by which glucocorticoids disrupt calcium homeostasis is unknown. Through gene expression array analysis, we found that dexamethasone induces a striking elevation of inositol 1,4,5-trisphosphate receptor (IP(3)R) levels in two murine lymphoma cell lines, WEHI7.2 and S49.A2. IP(3)R elevation was confirmed at both mRNA and protein levels. However, there was not a strong correlation between IP(3)R elevation and altered calcium homeostasis in terms of either kinetics or dose response. Moreover, IP(3)R knockdown, by either antisense or small interfering RNA, did not prevent either calcium disruption or apoptosis. Finally, DT40 lymphoma cells lacking all three IP(3)R isoforms were just as sensitive to dexamethasone-induced apoptosis as wild-type DT40 cells expressing all three IP(3)R isoforms. Thus, although alterations in intracellular calcium homeostasis contribute to glucocorticoid-induced apoptosis, these calcium alterations are not directly attributable to IP(3)R elevation.     

Flow cytometric detection of the mitochondrial BCL-2 protein in normal and neoplastic human lymphoid cells.

The bcl-2 proto-oncogene, rearranged and deregulated in B-cell lymphomas bearing the t(14;18) translocation, encodes an inner mitochondrial membrane protein that blocks apoptotic cell death. We have developed a sensitive immunofluorescence assay for the single- and multicolor flow cytometric analysis of bcl-2 protein in relation to other markers and cell cycle, based on a fixation-permeation step of cells with paraformaldehyde and Triton X100 and the use of a bcl-2 specific monoclonal antibody (MoAb). As an application of this method, we have examined the expression of bcl-2 in normal and neoplastic lymphoid cells. We have found that greater than 80% of normal T-and B-cells are bcl-2 positive; following in vitro mitogen activation, the bcl-2 reactivity decreased slightly in the former but markedly in latter cells. In both cases the bcl-2 expression was not restricted to a specific phase of the cell cycle, as evidenced by two-color analysis. On lymphoblastoid cell lines, the bcl-2 staining intensity was variable and not necessarily correlated to molecular rearrangements of the bcl-2 gene. Among fresh B-cell non-Hodgkin's lymphomas (B-NHL), most sporadic Burkitt's cases were bcl-2 negative. Of four centroblastic-centrocytic cases with rearrangements of the bcl-2 gene, only two presented elevated amounts of bcl-2 protein, indicating that the levels of bcl-2 are not diagnostic of the translocation.(ABSTRACT TRUNCATED AT 250 WORDS)     

IgG Fc receptor-bearing cells during early lymphoid cell development in the chicken

Cells from intraembryonic mesenchyme, yolk sac, bursa of Fabricius, and thymus from chicken embryos at different stages of development were studied for the presence of IgG Fc receptors by EA-rosette formation and binding of heat-aggregated chicken IgG (agg IgG). Cells with Fc receptors were found in high frequency in the intraembryonic mesenchyme as early as on the third day of incubation, in the yolk sac on the 7th day, in the bursa on the 10th day, and in the thymus on the 16th day of embryonic development. In the bursa the number of agg IgG binding cells increased with the age of the embryo and remained high after hatching, whereas in the thymus the peak value (76%) was observed on the 16th embryonic day, and after hatching only about 10% of the cells expressed the agg IgG receptors. The results also suggest that the appearance of IgG Fc receptors precedes the expression of B-L (Ia-like) antigens and of cytoplasmic and surface immunoglobulins on early lymphoid cells of the chicken embryo.