CR Cells Research Articles

Specific Arrest of Cardiogenesis in Cultured Embryonic Stem Cells Lacking Cripto-1

The molecular events of cardiac lineage specification and differentiation are largely unknown. Here we describe the involvement of a growth factor with an EGF-like domain, Cripto-1 (Cr-1), in cardiac differentiation. During embryonic development, Cr-1 is expressed in the mouse blastocyst, primitive streak, and later is restricted to the developing heart. To investigate the role of Cr-1, we have generated Cr-1-negative embryonic stem (ES) cell lines by homologous recombination. The resulting double “knockout” ES cells have selectively lost the ability to form beating cardiac myocytes, a process that can be rescued by reintroducing Cr-1 gene back into the Cr(−/−) cells. Furthermore, the lack of functional Cr-1 is correlated with absence of expression of cardiac-specific myosin light and heavy chain genes during differentiation. Differentiation into other cell types including skeletal muscle is not disrupted. These results suggest that Cr-1 is essential for contractile cardiomyocyte formationin vitro.

Correlation of poinsettia graft union development with transmission of the free-branching characteristic

Euphorbia pulcherrima Willd. ex. Klotzsch cv. ‘Eckespoint C-1 Red’ (CR), a restricted-branching poinsettia, and TR, a free-branching poinsettia, were approach grafted. Graft unions were removed for anatomical study at 0, 5, 10, 15, 20, 25 or 30 days after grafting, and the portion below the graft union was allowed to regrow. CR and TR plants had not yet adhered at 5 days after grafting. By 10 days after grafting, CR and TR parenchyma cells were actively dividing, producing new parenchyma cells (callus) on both sides of the necrotic layer. Parenchyma cells differentiated into nodules for the formation of new cambium by 25 days after grafting. TR and CR plants were not connected by new vascular tissue until 30 days after grafting. However, CR plants showed increased branching (axillary shoot development on lowermost nodes) by 10 days after grafting, indicating that vascular connections were not needed for transmission of the free-branching agent, which has been tentatively identified as a phytoplasma. Callus connecting TR and CR stems allowed transmission of the phytoplasma through the graft union.

Development of resistance to 9-nitro-camptothecin by human leukemia U-937 cells in vitro correlates with altered sensitivities to several anticancer drugs.

We have recently reported that exposure of human leukemia U-937 cells to progressively increasing concentrations of 9-nitro-camptothecin (9NC) resulted in cell sublines exhibiting various levels of resistance to 9NC. Here, we report responses of wild-type (U-937/wt) and 9NC-resistant (U-937/CR) cells to various anticancer drugs used extensively in cancer chemotherapy. U-937/CR cells were more sensitive than U-937/wt cells to several commonly used drugs of diverse origin including the topoisomerase II-directed drugs amsacrine, etoposide and daunorubicin; the vinca alkaloid vincristine; and the antimetabolite methotrexate. No responses were induced by carmustine in either cell type, whereas similar responses were induced by cytarabine. The sensitivity to the drugs was investigated by monitoring cell proliferation, by determining cell cycle perturbations assessed by flow cytometry analysis of DNA content and by microscopy of stained cells. The results in this report indicate that development of 9NC resistance by the U-937 cells is accompanied by increased sensitivities to other anticancer drugs in vitro and very likely in vivo.

Identification of a mutant human topoisomerase I with intact catalytic activity and resistance to 9-nitro-camptothecin.

Human U-937 myeloid leukemia cells were selected for resistance to increasing concentrations of the camptothecin derivative, 9-nitro-20(S)camptothecin (9-NC). The isolated single cell clone, designated U-937/CR, was approximately 20-fold resistant to 9-NC. Analysis of topoisomerase I (topo I) gene expression in U-937/CR cells demonstrated similar mRNA levels as compared with U-937 cells. Immunoblotting with an anti-topo I serum revealed reactive proteins at 100, 75, and 67 kDa which were expressed at the same level in the parental and 9-NC-resistant clones. These cell lines also demonstrated similar levels of topo I catalytic activity as determined by assaying nuclear extracts for relaxation of supercoiled plasmid DNA. In contrast, catalytic assays performed in the presence of 9-NC demonstrated that topo I activity from U-937/CR cells was approximately 10-fold more resistant than that from U-937 cells. Nucleotide sequencing of topo I cDNAs revealed the substitution of phenylalanine (TTC) at residue 361 in U-937 cells with serine (TCC) in the 9-NC-resistant clone. Expression and partial purification of the mutant topo I protein in Escherichia coli demonstrated resistance of this enzyme to 9-NC in catalytic assays. Taken together, these findings identify a novel mutation in topo I which confers resistance to 9-NC and support the involvement of this region in the interaction between topo I and 9-NC.

Effect of Pentazocine on the Cytotoxicity of Cortisone-Resistant Lymphocytes from Mouse Thymus

Pentazocine and its related compounds were examined for their effect on the cytotoxicity of cortisone-resistant lymphocytes (CR lymphocytes) against Ehrlich carcinoma cells. The following compounds were used: pentazocine, naloxone, levallorphan, eptazocine and morphine. CR lymphocytes were obtained from the thymus or spleens of mice injected i.p. with hydrocortisone acetate (125 mg/kg) 2 days before harvesting the lymphocytes. The mixture of tumor cells and CR lymphocytes was inoculated s.c. into mice after incubation in the presence or absence of 10 μM drugs. Five weeks after the inoculation, the percentage of mice developing a solid tumor among the recipients given the pentazocine-treated cell mixture of tumor cells and thymic CR lymphocytes was significantly smaller than the percentage in recipients given the cell mixture treated with or without other drugs (percent tumor takes: 21% and about 80%, respectively). Splenic CR lymphocytes did not show any cytotoxic effect, irrespective of the drug treatment. The pretreatment of CR lymphocytes or Ehrlich cells with 10 μM pentazocine did not affect the cytotoxicity of thymic and splenic CR lymphocytes. The proportion of the lymphocyte-conjugated tumor cells was significantly increased when the mixture of CR lymphocytes and tumor cells was incubated in the presence of pentazocine. The present results indicate that the cytotoxicity of thymic CR lymphocytes is enhanced by pentazocine possibly through the increase in the proportion of the lymphocyte-conjugated tumor cells but enhanced not by the other drugs.

Isolation and individual electrical stimulation of single smooth-muscle cells from the urinary bladder of the pig.

In contrast to striated muscle, measurements on strips of smooth muscle cannot be uniquely interpreted in terms of an array of contractile units. Therefore scaling down to the single-cell level is necessary to gain detailed understanding of the contractile process in this type of muscle. The present study describes the development of a method for isolating contractile single smooth muscle cells from pig urinary bladders. Contractile responses evoked by individual electrical stimulation were used as a measure of cell quality during development of the method. Responses were evaluated by measuring latency, contraction and relaxation times, as indicated by visible length changes, and stored on-line in a computer. Initial length, relative shortening and shortening speed were determined by measuring cell lengths in previously timed still video frames using a computer-controlled crosshair device. Increase of stimulus pulse duration resulted in improved responses, indicating that the observed shortening represented a physiological contractile response. Ultimately this method of evaluation was applied to two sets of cell preparations obtained by two different methods, one using only collagenase digestion, the other using mechanical manipulation as well. Both sets showed two main patterns of response to electrical stimulation: a pattern of contraction upon stimulation followed by enhanced contraction when stimulation was switched off (CK), and a pattern of contraction upon stimulation followed by relaxation when the stimulus was switched off (CR). The set of preparations containing the highest percentage of CR cells was found to be superior (i.e. greater initial length, shorter latency and contraction times, increased shortening and higher shortening speed). The method of isolation used for this set gives a high yield of contractile cells available for experimental use over a long span of time.

Gestational changes in the level of glucocorticoid-binding sites in fetal bovine cartilaginous tissues.

In order to assess glucocorticoid actions on fetal cartilage development, [3H]dexamethasone binding site levels in fetal bovine cartilaginous tissues from long bones were measured, using a whole cell assay at 37 degrees C. Displaceable [3H]dexamethasone binding in epiphysial growth cartilage was maximal (16.2 fmol/10(6) cells) in fetuses of 10-15 cm crown-rump length (CR), and declined to 22% of the maximum in fetuses of 20-30 cm CR. Subsequently, [3H]dexamethasone binding rose to a plateau (13.0 fmol/10(6) cells) in fetuses of 30-80 cm CR and declined in those of 80-100 cm CR. When measured in growth plate cartilage, [3H]dexamethasone binding was significantly higher in fetuses of 40-80 cm CR (39 fmol/10(6) cells) than in those of 80-100 cm CR. There was no significant change of [3H]dexamethasone binding affinities in epiphysial chondrocytes of 5-100 cm CR fetuses or in growth plate chondrocytes of 40-100 cm CR fetuses. These results demonstrate that fetal cartilaginous tissues during development possess varying cellular levels of glucocorticoid binding and may thus have temporal changes in sensitivity to glucocorticoid hormones.

Cytotoxicity of cortisone-resistant lymphoid cells from mice treated with adjuvants.

The cortisone-resistant lymphoid cells (CR cells) from mice treated with and without adjuvants were examined for their cytotoxicity on Ehrlich carcinoma cells (EC cells) or sarcoma-180 cells by the Winn test. Female mice of ddY strain were injected s.c. with Freund’s complete adjuvant (FCA), lipopolysaccharide (LPS) from E.coli or a group A hemolytic streptococcus (Cocci), and they were killed 14 days later. Using Hanks balanced salt solution, CR cells were prepared from thymus, spleen or mesenteric lymph node of mice injected i.p. with hydrocortisone acetate (125 mg/kg) 2 days before killing. CR cells were mixed with tumor cells (tumor cells/CR cells: 1/10), and the cell mixture was inoculated s.c. into mice (106 tumor cells/mouse). When EC cells were mixed with the mesenteric CR cells from untreated mice, 9 out of 20 mice given the cell mixture developed solid tumor. However, no tumor growth was observed in groups of mice receiving the mixture of EC cells and the mesenteric CR cells from animals treated with FCA or Cocci (0/20). In groups of mice given the mixture of EC cells and the thymic CR cells, the tumor incidence was 15/20 for CR cells from untreated mice and 6-7/20 for CR cells from mice treated with FCA or Cocci. On the other hand, the tumor growth was observed in almost all mice given the mixture of sarcoma-180 cells and the thymic or mesenteric CR cells form mice treated with FCA or Cocci.

IgG-Fc and C3 receptors in the chicken: distribution, tissue localization and functional significance

This paper summarizes data obtained in our laboratory on the demonstration of receptors for IgG-Fc (FcR) and complement (CR) on mononuclear cells of the fowl. A clearcut distinction of these two structures on spleen cells was achieved in rosette assays using SRBC coated with rabbit IgG as indicator cells which bind avian complement, but are not bound by the FcR. The tissue distribution and localization of FcR and CR positive cells was studied in mixed hemadsorption assays on sections of both central (bursa, thymus) and peripheral (spleen) lymphoid organs from normal chickens, as well as lymphocytic infiltrated thyroid glands from animals of the Obese strain (OS) afflicted with spontaneous autoimmune thyroiditis. The possible significance of both receptors in the pathogenesis of this autoimmune process is discussed.

Tissue localization of lymphocyte surface antigens and receptors for immunoglobulin G Fc and complement in the chicken.

Frozen sections of chicken lymphoid organs were examined for lymphocyte surface antigens by antisera to T and B lymphocytes (ATS;ABS), and for the presence of immunoglobulin (Ig) G Fc and complement receptors (FcR;CR) by hemadsorption with sheep erythrocytes (E) coated with chicken IgG(EA), and E coated with rabbit IgG and chicken complement (EAC). In the spleen FcR positive cells were confined to the periellipsoidal sheaths and the germinal centers. CR positive cells were found in the same spleen areas, as well as in the medulla of bursal follicles. These lymphoid areas reacted strongly with ABS, but they also stained with neutral alpha-naphthyl butyrate esterase, and phagocytosed carbon particles were found in the periellipsoidal sheaths. Furthermore, in vivo treatment with cyclophosphamide, which resulted in pronounced B-lymphocyte depletion, did not affect FcR activity, but reduced CR activity significantly. These data indicate that the FcR activity demonstrated in tissue sections is mainly confined to mononuclear phagocytes, while the CR positive cells are mainly B lymphocytes.

LH-stimulable adenylyl cyclase activity during the ovulatory cycle in granulosa cells of the three largest follicles and the postovulatory follicle of the domestic hen (Gallus domesticus).

The adenylyl cyclase (AC) activity in the granulosa (Gr) cells of the largest (F1), next largest (F2), third largest (F3), and postovulatory (POF) follicles throughout the ovulatory cycle of the domestic hen was measured. Granulosa cells were isolated at 24, 18, 12, 8, 6, 4, and 2 h before ovulation and basal and LH-stimulable AC activities were determined. There was a significant difference (P<0.05) in response between the basal and LH-stimulable activities of the F1 Gr at all times studied, with the most dramatic difference observed between 18 and 12 h before ovulation (P<0.001) (18 h, 58.42 ± 4.72 vs 12 h, 105.13 ± 4.89 pmoles cAMP/min/mg protein). There was a transient decrease in basal and LH-stimulable AC activities after the LH surge, followed by a significant (P<0.05) increase in basal activity 2 h before ovulation (6 h, 34.55 ± 3.94 vs 2 h, 53.76 ± 4.35 pmoles cAMP/min/mg protein). The fluctuations in basal and LH-stimulable AC activities of the F2 Gr cells were essentially the same as those of the F1 Gr except that the relative LH stimulation was less. In contrast, the pattern of AC activity of the F3 Gr cells differed from that of the F1 and F2 follicles. At all times assayed except 6 and 4 h before ovulation, the LH-stimulable activity was significantly greater (P<0.05) than basal values, which remained the same throughout the cycle. The LH-stimulable AC activity of the Gr cells of the POF was still significantly (P<0.001) elevated 2 h after ovulation, but declined to basal levels 4 h later. There was a significant increase in the basal AC activity of the POF at 24 h after ovulation. These results demonstrate that there is an increase in the cAMP synthesizing capacity of the Gr cells of preovulatory follicles as they approach ovulation. The loss of LH-stimulable AC activity in the POF Cr cells may be due to gonadotropin desensitization or cell death.

Target cell subpopulations for human thymic epithelial conditioned medium in the mouse thymus

Human thymic epithelial conditioned medium (HTECM) increases the reactivity of human and mouse thymocytes to Con A and PHA, and the reactivity of mouse thymocytes in mixed lymphocyte culture and in in vivo graft versus host reaction. In this study target cells for HTECM in subpopulations of mouse thymocytes at various times after hydrocortisone (HC) treatment were investigated. The PHA but not the Con A response of cortisone-resistant thymocytes (T cr, Day 3 after HC) was augmented by HTECM. Twelve days after HC treatment the thymus contained a population of cells (T 12) unresponsive to Con A and PHA and unresponsive to HTECM. This situation remained until Day 17. Between Days 17 and 19 the mitogen response was still very low but HTECM induced a marked increase in mitogen reactivity. After Day 19 the responses gradually returned to normal values. Histologically 12 days after HC the thymus corticomedullary differentiation was completely effaced and it contained an almost homogenous population of immature lymphoid cells. Histology returned to normal 17 days after HC. In the presence of T cr or lymph node (LN) cells, T 12 could be stimulated by mitogens. This response of mixtures of T 12 and T cr or LN cells to mitogen was increased by HTECM. The addition of mitomycin-treated T cr also resulted in an increase in the mitogen reactivity of T 12 by HTECM. From these data we conclude that T 12, although very immature, are sensitive to HTECM but regulatory T cells are needed to show this effect as measured by thymidine incorporation.

Trapped-electron doping of photovoltaic sandwich cells containing microcrystalline chlorophyll a

Cr‖Chla‖Hg sandwich cells with microcrystalline chlorophyll a exhibit rectifying I-V characteristics, whereas Au‖Chla‖Hg cells show Ohmic behavior. We present a comparative study of their ac and dc electrical properties. The impedances of both structures increase with age. The aging process can be reversed by light, suggesting that a trapped space charge controls the impedance levels. The results are consistent with an interpretation that the space charge is trapped electrons which function like a dopant in the Chl layer. The results with the Cr cells show the presence of an interfacial barrier region at the Cr-Chl contact which is responsible for the photovoltaic activity of these cells. The extent of this region is governed by the negative space charge. In a cell with approximately 7×1016 electrons/cm3, the width of the barrier is about 450 Å.

Effect of some nitrogen mustards and thoracic duct drainage on lymphocyte distribution in rats.

Distribution of &lt;sup&gt;51&lt;/sup&gt;Cr-tagged lymphocytes between lymph nodes and other tissues in rats was studied after an intravenous injection of labeled isogenic cells. The proportion of radioactivity in the lymph nodes was reduced by the following measures: heating the cells at 65 °C for 15 min, incubating them &lt;i&gt;in vitro &lt;/i&gt;with mechlorethamine (HN&lt;sub&gt;2&lt;/sub&gt;) or mannomustine; pretreating the cell donor animals with cyclophosphamide 30 mg/kg daily for 3 days. Incubating &lt;sup&gt;51&lt;/sup&gt;Cr-labeled cells with various bioeffective cyclophosphamide metabolites &lt;i&gt;in vitro &lt;/i&gt;had remarkably little effect on their subsequent distribution &lt;i&gt;in vivo &lt;/i&gt;in contrast to HN&lt;sub&gt;2&lt;/sub&gt;. Continually infusing cyclophosphamide for 24 h or a single massive infusion of cortisol hemisuccinate into recipients of &lt;sup&gt;51&lt;/sup&gt;Cr cells did not alter cell homing to lymphoid tissues.

Development of antigens in human cells infected with Simian virus 40.

Bissett, Marjorie L. (University of Michigan, Ann Arbor), and Francis E. Payne. Development of antigens in human cells infected with simian virus 40. J. Bacteriol. 91:743-749. 1966.-An explanation for the apparent infrequency with which human cells transform in response to exposure to simian virus 40 (SV40) was sought by following the development of virus-induced antigens in human euploid cells, strain CR. For about 8 weeks after exposure to a high multiplicity of SV40, only a small proportion of the cells produced tumor (T) or viral (V) antigen detected by immunofluorescence. Double-tracer staining techniques revealed that the development of T and V antigen in about 1% of the CR cells resembled that in green monkey kidney cells, strain BS-C-1, in which SV40 replicates and destroys all the cells. T antigen was detected before V antigen; both antigens were detected in the nucleus, but only V antigen appeared later in the cytoplasm. All intact cells that contained V antigen also contained T antigen. Infected CR cell cultures, before and after transformation or when in "crisis," contained only 0.1 to 1.0% of cells with both V and T antigen. Some CR cells contained only T antigen, and by 8 days after exposure to virus these cells were present as loose foci associated with an occasional cell containing V antigen. The proportion of CR cells with only T antigen increased from about 1% during the first 4 weeks to 8% at 7 weeks, and to nearly 100% at 11 weeks, when essentially all of the cells were epithelioid. Foci of epithelioid cells were first recognized in the 9th week. It was concluded that those CR cells that contained T antigen at any given time represented (i) a few cells that subsequently produced V antigen and lysed, and (ii) a progressively increasing population that produced only T antigen. If the latter population, in whole or in part, gave rise to the epithelioid transformed cells, then its initial size could account, at least in part, for the apparent infrequency with which human cells transform in response to SV40.