We have purified intact pp60v-src, the product of the Rous sarcoma virus src gene, over 2400-fold... more We have purified intact pp60v-src, the product of the Rous sarcoma virus src gene, over 2400-fold, based on the phosphorylation of tumor-bearing rabbit IgG. The purification procedure involved detergent extraction of the particulate fraction of the cells and sequential chromatography on hydroxylapatite, butyl agarose, DEAE-Sephacel, ADP-agarose, and Sephacryl S-200. Analysis of the preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single silver-stained band with an apparent molecular weight of 60,000. Our results show that the activities of this preparation were qualitatively similar to those described previously for partially purified pp60v-src. Upon analysis by two-dimensional gel electrophoresis, the purified pp60v-src yielded one major species which migrated to the same position as the least acidic of the three major species detectable in cellular lysates, suggesting that the pp60v-src had been dephosphorylated during the purification procedure. We found that pp60v-src was very prone to aggregation; to maintain it as a monomer both Nonidet P-40 and KCl were required. Under conditions which maintained pp60v-src as a monomer, the rate of autophosphorylation was independent of its concentration and thus proceeded via an intramolecular process. Preincubation of pp60v-src with ATP or GTP as well as nonphosphorylating analogs of ATP or GTP preserved its phosphorylating activity toward alpha-casein whereas its activity was reduced 80% upon preincubation in the absence of nucleotides. We suggest that protection with nucleotides rather than autophosphorylation accounts for the apparent increase in the activity of pp60v-src after incubation of the enzyme with ATP.
Removal of the lower epidermis from a tobacco leaf allows a faster and wider range of water fluxe... more Removal of the lower epidermis from a tobacco leaf allows a faster and wider range of water fluxes, without damaging the mesophyll. It also permits a more direct examination of the photosynthetic potential of the tissue at various levels of hydration. The rehydration rate of leaf discs is essentially linear. It decreases with leaf age and is correlated with the rate of dehydration, but it is independent of the tissue's water potential, as estimated by the isopiestic method. The hydraulic permeability coefficient of water influx is directly related to water potential of the tissue, suggesting a mechanism for the regulaton of the hydration level of the leaf tissue. The "energy of activation" of rehydration amounts to about 9 kilocalories per mole at intermediate dehydration, but it greatly declines following water loss in excess of 600 milligrams per gram fresh weight. The excessive dehydration is also characterized by a major increase in permeability (monitored by efflux of ions and materials absorbing ultraviolet light) and by a parallel decrease in photosynthetic activity. The interrelationship of these effects of excessive dehydration is discussed.
Protein kinase activity in incubated liver slices from 35 degrees C heat-acclimated (HA) hamsters... more Protein kinase activity in incubated liver slices from 35 degrees C heat-acclimated (HA) hamsters was 70% higher than in similar slices from 23 degrees C control (C) hamsters. Adding glucagon to the incubation medium increased protein kinase activity by 65% in slices from C animals, but by only 30% in slices from HA animals. Binding of [3H]cAMP to proteins of a low-speed supernatant fraction of incubated and homogenized slices was 30% lower for HA than for C hamsters. For each acclimation group this binding was reduced 30% by incubation of the slices with glucagon. The activities of phosphorylase kinase, phosphorylase phosphatase, and phosphorylase alpha in slices incubated with or without glucagon did not differ between groups. Addition of glucagon increased phosphorylase kinase by 30% and phosphorylase alpha by 40% but caused no change in phosphorylase phosphatase activity. These results suggest that heat acclimation of the hamster increases the amount of a species of liver protein kinase that is different from the one that mediates the effect of glucagon on glycogenolysis.
American Journal of Physiology-cell Physiology, Oct 1, 1993
Arginine vasopressin (AVP) has been shown to stimulate tyrosine phosphorylation and activation of... more Arginine vasopressin (AVP) has been shown to stimulate tyrosine phosphorylation and activation of p42 mitogen-activated protein (MAP) kinase (p42MAPK) in vascular smooth muscle cells (VSMC). In VSMC, AVP increases free intracellular Ca2+ concentration ([Ca2+]i) and activates protein kinase C (PKC) through activation of phospholipase C. The contribution of PKC and [Ca2+]i in p42MAPK regulation was therefore determined. Activation of PKC by phorbol 12-myristate 13-acetate (PMA) stimulated tyrosine phosphorylation and activation of p42MAPK to the same extent as AVP. Inhibition of PKC by staurosporine or downregulation of PKC by PMA pretreatment abolished AVP-induced stimulation of p42MAPK. When [Ca2+]i was elevated to the same level as with AVP, using either ionomycin (0.1 microM) or thapsigargin (0.1 microM), MAP kinase was only partially activated. Elevation of [Ca2+]i to supraphysiological levels by 1 microM ionomycin stimulated MAP kinase activity to the same extent as AVP. This effect was blocked by downregulation of PKC. The intracellular Ca2+ chelator BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] blocked AVP-induced [Ca2+]i increase but did not affect AVP stimulation of p42MAPK. Thus AVP-induced activation of p42MAPK requires only the activation of PKC but not an increase in [Ca2+]i.
This report describes quantitative results of in vitro phosphorylation of the Rous sarcoma virus ... more This report describes quantitative results of in vitro phosphorylation of the Rous sarcoma virus transforming gene product, pp60src, using ATP or GTP as phosphate donors. The Km values for the phosphorylation of pp60src by ATP and GTP were similar (10-36 and 25-36 microM, respectively) and the Vmax values were different (5-7 and 1.5-1.7 nmol min-1 mg-1 of pp60src, respectively). The radiolabeling of pp60src by [gamma-32P] ATP was inhibited by ADP and dATP at 20-fold higher concentrations by 75 and 83%, respectively. Other nucleotides served as weaker inhibitors under the same conditions. The radiolabeling of pp60src by [gamma-32P]GTP had lower specificity for this nucleotide, since ATP, dATP, ADP, dGTP, GDP, and TTP had at least a 50% inhibitory effect. The phosphorylated products of approximate Mr = 60,000 that were produced with ATP or GTP were shown to be the same protein molecule since they both could be immunoprecipitated with antibody raised against p60src produced by bacterial recombinants. Structural analysis revealed that the use of GTP resulted in phosphorylation of a tyrosine residue on the COOH-terminal region of pp60src, apparently the same site which contains the tyrosine phosphorylated in infected cells. In contrast, the use of ATP resulted in phosphorylation of several additional tyrosine residues on the NH2-terminal region of the molecule. In thermolability studies, the t1/2 values for the phosphorylation of pp66src in preparations from wild type virus-infected chicken cells were 5.1 min for both ATP and GTP, whereas the t1/2 values for the phosphorylation of pp60src in preparations from temperature-sensitive transformation mutant-infected cells were 1.1 min for both phosphate donors. Similar observations were found with alpha-casein as substrate.
The International Journal of Biochemistry & Cell Biology, 2008
Human bone marrow mesenchymal stem cells are multipotent cells with enormous potential for cellul... more Human bone marrow mesenchymal stem cells are multipotent cells with enormous potential for cellular therapies. Identifying those mediators that induce human bone marrow mesenchymal stem cell proliferation and elucidating the signaling networks involved will encourage clinical efforts exploiting such cells. Here, we demonstrate that platelet-derived growth factor-BB and basic fibroblast growth factor induce human bone marrow mesenchymal stem cell proliferation. Platelet-derived growth factor-BB induced human bone marrow mesenchymal stem cell proliferation via complete activation of the Janus-activated kinase-signal transducers and activators of transcription cascade, inducing signal transducer and activator of transcription 3 tyrosine and serine phosphorylation as well as Janus-activated kinase 2 tyrosine phosphorylation. Janus-activated kinase 2 was required for signal transducer and activator of transcription 3 tyrosine phosphorylation, whereas the extracellular signal-regulated kinase 1/2 mediated signal transducer and activator of transcription 3 serine phosphorylation in response to platelet-derived growth factor-BB. Furthermore, platelet-derived growth factor-BB was shown to promote nuclear translocation of signal transducer and activator of transcription 3. By contrast, basic fibroblast growth factor-stimulated human bone marrow mesenchymal stem cell proliferation was mediated via the extracellular signal-regulated kinase 1/2 pathway without involvement of the Janus-activated kinase-signal transducers and activators of transcription cascade. Importantly, platelet-derived growth factor-BB and basic fibroblast growth factor induced human bone marrow mesenchymal stem cell proliferation without affecting their osteogenic differentiation potential. Together, our study highlights the role of several growth factors in human bone marrow mesenchymal stem cell proliferation and the signaling pathways involved in the process. This information is crucial for achieving a better control over the human bone marrow mesenchymal stem cell expansion process.
Hepoxilin-A(3) (Hx-A(3)) is produced by platelets in response to shear-stress. It has an antithro... more Hepoxilin-A(3) (Hx-A(3)) is produced by platelets in response to shear-stress. It has an antithrombotic effect on platelets. A low Hx-A(3) level may contribute to the high thrombogenic state that exists in patients with acute coronary syndromes. Since we have previously demonstrated that the regulatory volume decrease (RVD) of human platelets exposed to hypotonic solutions is controlled by Hx-A(3) it is possible that the RVD rate reflects Hx-A(3) activity. In this study, the RVD rate of platelets taken from a healthy control group (n=21) was compared to that of patients with chronic ischemic heart disease (n=23), acute ischemic heart disease (n = 24) and acute myocardial infarction (MI, n = 29). The RVD rate of the control group was significantly higher than the other three groups (P < 0.001). The addition of 100 nM of Hx-A, to the platelets of eight patients with MI increased their RVD rate to that of the controls. Patients with diabetes mellitus or hypertension have the lowest RVD rates. Medications such as aspirin, heparin, and streptokinase did not affect the Hx-A(3) activity of platelets obtained from patients with ischemic heart disease. The results of the present study indicate that patients with acute ischemia may have a low level of platelet Hx-A(3) activity. This possible low level of Hx-A, activity may be associated with a failure to develop an antithrombotic reaction to the shear-stress forces generated during acute ischemia.
We have previously shown that a 2-chloro-1,4-naphthoquinone derivative (TW-92) induces cell death... more We have previously shown that a 2-chloro-1,4-naphthoquinone derivative (TW-92) induces cell death in leukemia cells. TW-92 exhibited relatively high selectivity towards primary Acute Myeloid Leukemia (AML) cells, as compared to normal mononuclear cells. In view of the selectivity of this family of naphthoquinones, novel chloroaminophenylnaphthoquinone isomers with different methyl substitutions on the phenyl ring were synthesized, and their effect on leukemia cells was tested. These compounds induced cell death in U937 human myeloid leukemia cells, which was prominent following 48 h of culture. Structure-activity relationship studies revealed that TW-74, a novel chloronaphthoquinone with a methyl group at the meta (m) position, was the most active derivative in inducing apoptosis. The mechanism underlying cell death induction by TW-74 was further investigated in U937 cells, a monocytic cell line which serves as a sensitive model of apoptosis induction. TW-74 induced rapid activation...
Background —The myocardium is unable to regenerate because cardiomyocytes cannot replicate after ... more Background —The myocardium is unable to regenerate because cardiomyocytes cannot replicate after injury. The heart is therefore an attractive target for tissue engineering to replace infarcted myocardium and enhance cardiac function. We tested the feasibility of bioengineering cardiac tissue within novel 3-dimensional (3D) scaffolds. Methods and Results —We isolated and grew fetal cardiac cells within 3D porous alginate scaffolds. The cell constructs were cultured for 4 days to evaluate viability and morphology before implantation. Light microscopy revealed that within 2 to 3 days in culture, the dissociated cardiac cells form distinctive, multicellular contracting aggregates within the scaffold pores. Seven days after myocardial infarction, rats were randomized to biograft implantation (n=6) or sham-operation (n=6) into the myocardial scar. Echocardiography study was performed before and 65±5 days after implantation to assess left ventricular (LV) remodeling and function. Hearts we...
As a model system to study parvoviral oncosuppression, Ehrlich ascites (EA) cells were injected i... more As a model system to study parvoviral oncosuppression, Ehrlich ascites (EA) cells were injected into the peritoneal cavities of ICR mice, and the effect of im injection of minute virus of mice (MVM) on EA tumor growth was examined. Coinjection with MVM resulted in a dramatic inhibition of EA tumor formation. Tumor suppression required viable, infectious virus. Mice that had survived one EA-MVM coinjection acquired long-term resistance to additional injections of EA cells.
We have purified intact pp60v-src, the product of the Rous sarcoma virus src gene, over 2400-fold... more We have purified intact pp60v-src, the product of the Rous sarcoma virus src gene, over 2400-fold, based on the phosphorylation of tumor-bearing rabbit IgG. The purification procedure involved detergent extraction of the particulate fraction of the cells and sequential chromatography on hydroxylapatite, butyl agarose, DEAE-Sephacel, ADP-agarose, and Sephacryl S-200. Analysis of the preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single silver-stained band with an apparent molecular weight of 60,000. Our results show that the activities of this preparation were qualitatively similar to those described previously for partially purified pp60v-src. Upon analysis by two-dimensional gel electrophoresis, the purified pp60v-src yielded one major species which migrated to the same position as the least acidic of the three major species detectable in cellular lysates, suggesting that the pp60v-src had been dephosphorylated during the purification procedure. We found that pp60v-src was very prone to aggregation; to maintain it as a monomer both Nonidet P-40 and KCl were required. Under conditions which maintained pp60v-src as a monomer, the rate of autophosphorylation was independent of its concentration and thus proceeded via an intramolecular process. Preincubation of pp60v-src with ATP or GTP as well as nonphosphorylating analogs of ATP or GTP preserved its phosphorylating activity toward alpha-casein whereas its activity was reduced 80% upon preincubation in the absence of nucleotides. We suggest that protection with nucleotides rather than autophosphorylation accounts for the apparent increase in the activity of pp60v-src after incubation of the enzyme with ATP.
Removal of the lower epidermis from a tobacco leaf allows a faster and wider range of water fluxe... more Removal of the lower epidermis from a tobacco leaf allows a faster and wider range of water fluxes, without damaging the mesophyll. It also permits a more direct examination of the photosynthetic potential of the tissue at various levels of hydration. The rehydration rate of leaf discs is essentially linear. It decreases with leaf age and is correlated with the rate of dehydration, but it is independent of the tissue's water potential, as estimated by the isopiestic method. The hydraulic permeability coefficient of water influx is directly related to water potential of the tissue, suggesting a mechanism for the regulaton of the hydration level of the leaf tissue. The "energy of activation" of rehydration amounts to about 9 kilocalories per mole at intermediate dehydration, but it greatly declines following water loss in excess of 600 milligrams per gram fresh weight. The excessive dehydration is also characterized by a major increase in permeability (monitored by efflux of ions and materials absorbing ultraviolet light) and by a parallel decrease in photosynthetic activity. The interrelationship of these effects of excessive dehydration is discussed.
Protein kinase activity in incubated liver slices from 35 degrees C heat-acclimated (HA) hamsters... more Protein kinase activity in incubated liver slices from 35 degrees C heat-acclimated (HA) hamsters was 70% higher than in similar slices from 23 degrees C control (C) hamsters. Adding glucagon to the incubation medium increased protein kinase activity by 65% in slices from C animals, but by only 30% in slices from HA animals. Binding of [3H]cAMP to proteins of a low-speed supernatant fraction of incubated and homogenized slices was 30% lower for HA than for C hamsters. For each acclimation group this binding was reduced 30% by incubation of the slices with glucagon. The activities of phosphorylase kinase, phosphorylase phosphatase, and phosphorylase alpha in slices incubated with or without glucagon did not differ between groups. Addition of glucagon increased phosphorylase kinase by 30% and phosphorylase alpha by 40% but caused no change in phosphorylase phosphatase activity. These results suggest that heat acclimation of the hamster increases the amount of a species of liver protein kinase that is different from the one that mediates the effect of glucagon on glycogenolysis.
American Journal of Physiology-cell Physiology, Oct 1, 1993
Arginine vasopressin (AVP) has been shown to stimulate tyrosine phosphorylation and activation of... more Arginine vasopressin (AVP) has been shown to stimulate tyrosine phosphorylation and activation of p42 mitogen-activated protein (MAP) kinase (p42MAPK) in vascular smooth muscle cells (VSMC). In VSMC, AVP increases free intracellular Ca2+ concentration ([Ca2+]i) and activates protein kinase C (PKC) through activation of phospholipase C. The contribution of PKC and [Ca2+]i in p42MAPK regulation was therefore determined. Activation of PKC by phorbol 12-myristate 13-acetate (PMA) stimulated tyrosine phosphorylation and activation of p42MAPK to the same extent as AVP. Inhibition of PKC by staurosporine or downregulation of PKC by PMA pretreatment abolished AVP-induced stimulation of p42MAPK. When [Ca2+]i was elevated to the same level as with AVP, using either ionomycin (0.1 microM) or thapsigargin (0.1 microM), MAP kinase was only partially activated. Elevation of [Ca2+]i to supraphysiological levels by 1 microM ionomycin stimulated MAP kinase activity to the same extent as AVP. This effect was blocked by downregulation of PKC. The intracellular Ca2+ chelator BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid] blocked AVP-induced [Ca2+]i increase but did not affect AVP stimulation of p42MAPK. Thus AVP-induced activation of p42MAPK requires only the activation of PKC but not an increase in [Ca2+]i.
This report describes quantitative results of in vitro phosphorylation of the Rous sarcoma virus ... more This report describes quantitative results of in vitro phosphorylation of the Rous sarcoma virus transforming gene product, pp60src, using ATP or GTP as phosphate donors. The Km values for the phosphorylation of pp60src by ATP and GTP were similar (10-36 and 25-36 microM, respectively) and the Vmax values were different (5-7 and 1.5-1.7 nmol min-1 mg-1 of pp60src, respectively). The radiolabeling of pp60src by [gamma-32P] ATP was inhibited by ADP and dATP at 20-fold higher concentrations by 75 and 83%, respectively. Other nucleotides served as weaker inhibitors under the same conditions. The radiolabeling of pp60src by [gamma-32P]GTP had lower specificity for this nucleotide, since ATP, dATP, ADP, dGTP, GDP, and TTP had at least a 50% inhibitory effect. The phosphorylated products of approximate Mr = 60,000 that were produced with ATP or GTP were shown to be the same protein molecule since they both could be immunoprecipitated with antibody raised against p60src produced by bacterial recombinants. Structural analysis revealed that the use of GTP resulted in phosphorylation of a tyrosine residue on the COOH-terminal region of pp60src, apparently the same site which contains the tyrosine phosphorylated in infected cells. In contrast, the use of ATP resulted in phosphorylation of several additional tyrosine residues on the NH2-terminal region of the molecule. In thermolability studies, the t1/2 values for the phosphorylation of pp66src in preparations from wild type virus-infected chicken cells were 5.1 min for both ATP and GTP, whereas the t1/2 values for the phosphorylation of pp60src in preparations from temperature-sensitive transformation mutant-infected cells were 1.1 min for both phosphate donors. Similar observations were found with alpha-casein as substrate.
The International Journal of Biochemistry & Cell Biology, 2008
Human bone marrow mesenchymal stem cells are multipotent cells with enormous potential for cellul... more Human bone marrow mesenchymal stem cells are multipotent cells with enormous potential for cellular therapies. Identifying those mediators that induce human bone marrow mesenchymal stem cell proliferation and elucidating the signaling networks involved will encourage clinical efforts exploiting such cells. Here, we demonstrate that platelet-derived growth factor-BB and basic fibroblast growth factor induce human bone marrow mesenchymal stem cell proliferation. Platelet-derived growth factor-BB induced human bone marrow mesenchymal stem cell proliferation via complete activation of the Janus-activated kinase-signal transducers and activators of transcription cascade, inducing signal transducer and activator of transcription 3 tyrosine and serine phosphorylation as well as Janus-activated kinase 2 tyrosine phosphorylation. Janus-activated kinase 2 was required for signal transducer and activator of transcription 3 tyrosine phosphorylation, whereas the extracellular signal-regulated kinase 1/2 mediated signal transducer and activator of transcription 3 serine phosphorylation in response to platelet-derived growth factor-BB. Furthermore, platelet-derived growth factor-BB was shown to promote nuclear translocation of signal transducer and activator of transcription 3. By contrast, basic fibroblast growth factor-stimulated human bone marrow mesenchymal stem cell proliferation was mediated via the extracellular signal-regulated kinase 1/2 pathway without involvement of the Janus-activated kinase-signal transducers and activators of transcription cascade. Importantly, platelet-derived growth factor-BB and basic fibroblast growth factor induced human bone marrow mesenchymal stem cell proliferation without affecting their osteogenic differentiation potential. Together, our study highlights the role of several growth factors in human bone marrow mesenchymal stem cell proliferation and the signaling pathways involved in the process. This information is crucial for achieving a better control over the human bone marrow mesenchymal stem cell expansion process.
Hepoxilin-A(3) (Hx-A(3)) is produced by platelets in response to shear-stress. It has an antithro... more Hepoxilin-A(3) (Hx-A(3)) is produced by platelets in response to shear-stress. It has an antithrombotic effect on platelets. A low Hx-A(3) level may contribute to the high thrombogenic state that exists in patients with acute coronary syndromes. Since we have previously demonstrated that the regulatory volume decrease (RVD) of human platelets exposed to hypotonic solutions is controlled by Hx-A(3) it is possible that the RVD rate reflects Hx-A(3) activity. In this study, the RVD rate of platelets taken from a healthy control group (n=21) was compared to that of patients with chronic ischemic heart disease (n=23), acute ischemic heart disease (n = 24) and acute myocardial infarction (MI, n = 29). The RVD rate of the control group was significantly higher than the other three groups (P < 0.001). The addition of 100 nM of Hx-A, to the platelets of eight patients with MI increased their RVD rate to that of the controls. Patients with diabetes mellitus or hypertension have the lowest RVD rates. Medications such as aspirin, heparin, and streptokinase did not affect the Hx-A(3) activity of platelets obtained from patients with ischemic heart disease. The results of the present study indicate that patients with acute ischemia may have a low level of platelet Hx-A(3) activity. This possible low level of Hx-A, activity may be associated with a failure to develop an antithrombotic reaction to the shear-stress forces generated during acute ischemia.
We have previously shown that a 2-chloro-1,4-naphthoquinone derivative (TW-92) induces cell death... more We have previously shown that a 2-chloro-1,4-naphthoquinone derivative (TW-92) induces cell death in leukemia cells. TW-92 exhibited relatively high selectivity towards primary Acute Myeloid Leukemia (AML) cells, as compared to normal mononuclear cells. In view of the selectivity of this family of naphthoquinones, novel chloroaminophenylnaphthoquinone isomers with different methyl substitutions on the phenyl ring were synthesized, and their effect on leukemia cells was tested. These compounds induced cell death in U937 human myeloid leukemia cells, which was prominent following 48 h of culture. Structure-activity relationship studies revealed that TW-74, a novel chloronaphthoquinone with a methyl group at the meta (m) position, was the most active derivative in inducing apoptosis. The mechanism underlying cell death induction by TW-74 was further investigated in U937 cells, a monocytic cell line which serves as a sensitive model of apoptosis induction. TW-74 induced rapid activation...
Background —The myocardium is unable to regenerate because cardiomyocytes cannot replicate after ... more Background —The myocardium is unable to regenerate because cardiomyocytes cannot replicate after injury. The heart is therefore an attractive target for tissue engineering to replace infarcted myocardium and enhance cardiac function. We tested the feasibility of bioengineering cardiac tissue within novel 3-dimensional (3D) scaffolds. Methods and Results —We isolated and grew fetal cardiac cells within 3D porous alginate scaffolds. The cell constructs were cultured for 4 days to evaluate viability and morphology before implantation. Light microscopy revealed that within 2 to 3 days in culture, the dissociated cardiac cells form distinctive, multicellular contracting aggregates within the scaffold pores. Seven days after myocardial infarction, rats were randomized to biograft implantation (n=6) or sham-operation (n=6) into the myocardial scar. Echocardiography study was performed before and 65±5 days after implantation to assess left ventricular (LV) remodeling and function. Hearts we...
As a model system to study parvoviral oncosuppression, Ehrlich ascites (EA) cells were injected i... more As a model system to study parvoviral oncosuppression, Ehrlich ascites (EA) cells were injected into the peritoneal cavities of ICR mice, and the effect of im injection of minute virus of mice (MVM) on EA tumor growth was examined. Coinjection with MVM resulted in a dramatic inhibition of EA tumor formation. Tumor suppression required viable, infectious virus. Mice that had survived one EA-MVM coinjection acquired long-term resistance to additional injections of EA cells.
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