ABSTRACT
To determine effects of exposure of parental animals to antibiotics on antibiotic resistance in bacteria of offspring, sows were either treated or not treated with oxytetracycline prior to farrowing and their pigs were challenged with Salmonella enterica Typhimurium and treated or not treated with oxytetracycline and apramycin. Fecal Escherichia coli were obtained from sows, and E. coli and salmonella were recovered from pigs. Antibiotic resistance patterns of isolates were determined using a minimum inhibitory concentration (MIC) analysis. Polymerase chain reaction (PCR) and electroporation were used to characterize the genetic basis for the resistance and to determine the location of resistance genes. Treatments had little effect on resistance of the salmonella challenge organism. The greatest resistance to apramycin occurred in E. coli from pigs treated with apramycin and whose sows had earlier exposure to oxytetracycline. Resistance to oxytetracycline was consistently high throughout the study in isolates from all pigs and sows; however, greater resistance was noted in pigs nursing sows that had previous exposure to that drug. The aac(3)-IV gene, responsible for apramycin resistance, was found in approximately 90% of apramycin-resistant isolates and its location was determined to be on plasmids. Several resistant E. coli bio-types were found to contain the resistance gene. These results indicate that resistance to apramycin and oxytetracycline in E. coli of pigs is affected by previous use of oxytetracycline in sows.
Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli Infections/veterinary , Escherichia coli/drug effects , Salmonella Infections, Animal/microbiology , Salmonella typhimurium/drug effects , Swine Diseases/microbiology , Animals , Animals, Newborn , Animals, Suckling , Anti-Bacterial Agents/therapeutic use , Drug Resistance, Bacterial , Escherichia coli/isolation & purification , Escherichia coli Infections/drug therapy , Escherichia coli Infections/microbiology , Feces/microbiology , Female , Microbial Sensitivity Tests/veterinary , Nebramycin/analogs & derivatives , Nebramycin/pharmacology , Nebramycin/therapeutic use , Oxytetracycline/pharmacology , Oxytetracycline/therapeutic use , Pregnancy , Random Allocation , Salmonella Infections, Animal/drug therapy , Salmonella typhimurium/isolation & purification , Swine , Swine Diseases/drug therapyABSTRACT
The objective of this study was to understand factors responsible for apoptotic body formation and release during apoptosis. We have found that inhibition of mono-ADP ribosylation after ultraviolet (UV) light induction of apoptosis in HL-60 cells does not block caspase-3 activation, gelsolin cleavage, or endonucleolytic DNA fragmentation. However, the cytoskeletal features of apoptosis leading to apoptotic body formation and release were inhibited by meta-iodobenzylguanidine (MIBG) and novobiocin, potent inhibitors of arginine-specific mono-ADP-ribosyltransferases (mono-ADPRTs). Suppression of mono-ADP ribosylation as late as 120 min following UV irradiation blocked the depolymerization of actin and release of apoptotic bodies. This suggested that the cytoskeletal changes of apoptosis may be decoupled from the caspase cascade and that there may be a biochemical event either distal to or independent of caspase-3 that regulates apoptotic body formation. To test the hypothesis that ADP ribosylation of actin may occur with the induction of apoptosis, an in vivo assay of mono-ADPRT activity using an antibody against ADP-ribosylarginine was used. An approximately 64% increase in the ADP ribosylation of actin was observed at 2 h following exposure to UV light. When MIBG or novobiocin was present, the ADP ribosylation of actin was only 14-18% above the levels observed in control nonirradiated cells. The current study is the first to demonstrate a relationship between ADP-ribosylation of actin and the formation of apoptotic bodies.
Subject(s)
ADP Ribose Transferases/antagonists & inhibitors , Apoptosis/physiology , 3-Iodobenzylguanidine/pharmacology , Actins/analysis , Caspase 3 , Caspases , Endonucleases/metabolism , Gelsolin/metabolism , HL-60 Cells/radiation effects , Humans , Novobiocin/pharmacology , Protein Processing, Post-Translational , Ultraviolet RaysABSTRACT
Although sulfur mustard (SM) has been reported to be a DNA alkylating agent, it is not clear how much of the cytotoxicity of this agent is secondary to DNA damage. To test the hypothesis that the presence of a nucleus is required for the toxicity of sulfur mustard, enucleated endothelial cytoplasts were treated with SM. Using a combination of biochemical and microscopic assays, we demonstrate that some aspects of SM-induced cell death may be dependent on the presence of a nucleus, while others may not be. For example, it was found that cytoskeletal changes, such as loss of stress fibers and rounding, proceed in response to sulfur mustard treatment even in the absence of a nucleus. However, significant further increases in caspase activity and the associated phosphatidylserine translocation were not observed in cytoplasts treated with 500 microM SM for 6 h (following a 20-h recovery at the end of cytoplast preparation). In contrast, cytoplasts treated with chelerythrine, an agent previously reported to induce rapid apoptosis, demonstrated increases in caspase activity in cytoplasts comparable to that observed in the nucleated cells. This indicates that sulfur mustard-induced alkylation of nuclear DNA may be an important stimulus for activation of caspases in nucleated cells. Interestingly, the baseline caspase activity in cytoplasts was greater than in nucleated cells. Analysis of the time course of caspase activation in untreated adherent cytoplasts indicated that the activity increases initially and then stabilizes by 8 h to a low level that was comparable to the level observed at 26 h in untreated cytoplasts. This indicates that cytoplasts are able to tolerate stable low levels of caspase activity and not proceed immediately into the execution phase of apoptosis. The cytoplast model may be quite useful in the toxicological assessment of agents that are thought to exert their toxicity through DNA damage.
Subject(s)
Cell Nucleus/drug effects , Chemical Warfare Agents/toxicity , Mustard Gas/toxicity , Animals , Annexin A5/pharmacology , Caspase 3 , Caspases/metabolism , Cattle , Cell Death/drug effects , Cell Membrane/drug effects , Cell Membrane/ultrastructure , Cell Survival/drug effects , Fluoresceins , Microscopy, Fluorescence , Phosphatidylserines/metabolismABSTRACT
The relationship between aryl hydrocarbon receptor (AHR) content and susceptibility to apoptosis was examined in the murine hepatoma 1c1c7 cell line and a series of variants having different levels of AHR expression. Exposure of 1c1c7 cultures to N-acetylsphingosine (C2-ceramide) caused a concentration-dependent inhibition of cell proliferation, loss of viability, and induction of apoptosis as monitored by analyses of DNA fragmentation and caspase activation. A variant cell line (Tao) having approximately 10% of the AHR content of 1c1c7 cells also arrested following exposure to C2-ceramide, but did not undergo apoptosis. Modulation of 1c1c7 and Tao AHR contents by transfection of Ahr antisense and sense constructs, respectively, confirmed the relationship between AHR content and susceptibility to C2-ceramide-induced apoptosis. C2-ceramide also induced the apoptosis of an AHR-containing cell line lacking the aryl hydrocarbon receptor nuclear translocator protein. AHR ligands (i.e. 2,3,7,8-tetrachlorodibenzo-p-dioxin and alpha-naphthoflavone) neither induced apoptosis nor modulated the development of apoptosis in C2-ceramide-treated 1c1c7 cultures. AHR content did not affect staurosporine- or doxorubicin-induced apoptosis. These results suggest the AHR modulates aspects of ceramide signaling associated with the induction of apoptosis but not cell cycle arrest, and does so by a mechanism that is independent of its interaction with aryl hydrocarbon receptor nuclear translocator and exogenous AHR ligands.
Subject(s)
Apoptosis/drug effects , DNA-Binding Proteins , Liver Neoplasms, Experimental/pathology , Receptors, Aryl Hydrocarbon/physiology , Sphingosine/analogs & derivatives , Animals , Aryl Hydrocarbon Receptor Nuclear Translocator , Doxorubicin/pharmacology , Liver Neoplasms, Experimental/metabolism , Mice , Sphingosine/pharmacology , Staurosporine/pharmacology , Transcription Factors/physiology , Tumor Cells, CulturedABSTRACT
PD98059 [2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one] is a flavonoid and a potent inhibitor of mitogen-activated protein kinase kinase (MEK). Concentrations of PD98059 of =20 muM were not cytotoxic to cultures of the immortalized human breast epithelial cell line MCF10A. The agent was weakly cytostatic at concentrations of >/=10 microM. In vivo exposure of cultures to =20 microM PD98059 for 2-22 hr did not affect overall extracellular signal-regulated kinase contents; however, exposure to PD98059 resulted in a rapid loss (>95%) of the dually phosphorylated forms of extracellular signal-regulated kinase (IC50 = 1 muM). Treatment of cultures with PD98059 of >/=1 muM either at the time of addition or up to 48 hr before the addition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed in a concentration-dependent manner the accumulation of induced steady state CYP1A1, CYP1B1, and NQO1 mRNAs. The addition of PD98059 to rat liver cytosol just before the addition of TCDD suppressed TCDD binding (IC50 = 4 muM) and aryl hydrocarbon receptor (AHR) transformation (IC50 = 1 muM), as measured by sucrose gradient centrifugation and electrophoretic mobility shift assays. Flavone and flavanone, two closely related structural analogs of PD98059, inhibited AHR transformation by TCDD with IC50 values similar to that obtained with PD98059. However, neither analog was as potent as PD98059 in inhibiting MEK (IC50 approximately 190 muM for both). These results suggest that PD98059 is a ligand for the AHR and functions as an AHR antagonist at concentrations commonly used to inhibit MEK and signaling processes that entail MEK activation.
Subject(s)
Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Protein Kinase Inhibitors , Receptors, Aryl Hydrocarbon/antagonists & inhibitors , Animals , Binding, Competitive , Cell Line , DNA/metabolism , Humans , Mitogen-Activated Protein Kinase Kinases , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Nerve Tissue Proteins/antagonists & inhibitors , Polychlorinated Dibenzodioxins/metabolism , Polychlorinated Dibenzodioxins/pharmacology , Rats , Receptors, Aryl Hydrocarbon/physiology , Transcriptional Activation/drug effectsABSTRACT
The immortalized human epithelial cell line MCF10A has the phenotypic characteristics of normal breast cells. Exposure of MCF10A cultures to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) stimulated the transcriptional activation of cytochrome P450 1A1 (CYP1A1), and CYP1B1, and NAD(P)H:quinone oxidoreductase. Northern blot hybridization and nuclear run-on assays demonstrated that transcriptional activation of these genes was suppressed in stably transfected cultures expressing an Ha-ras oncogene (the MCF10A-NeoT line). Similar suppression did not occur in stably transfected lines carrying the expression vector or a normal c-Ha-ras protooncogene. Western blot analyses and immunofluorescence microscopy demonstrated that the lack of inducibility in MDF10A-NeoT cells reflected neither reductions in aryl hydrocarbon receptor (AHR) and aryl hydrocarbon nuclear translocator protein nor prevention of TCDD-induced AHR translocation to the nucleus. Suppression did correlate with reductions in DNA-AHR complex formation, as analyzed by gel retardation assays of soluble cell extracts treated in vitro with TCDD. The induction of Cyp1a-1 by TCDD was also analyzed in transgenic mice that expressed a v-Ha-ras oncogene exclusively in their keratinocytes. Relative to littermates lacking the transgene, the induction of Cyp1a-1 by TCDD was partially suppressed (about 50%) in the epidermises of v-Ha-ras-positive transgenic mice. However, normal levels of Cyp1a-1 induction occurred in the livers of the same mice. induction of Cyp1a-1 by TCDD was also suppressed (more than 98%) in chemically induced skin papillomas having Ha-ras mutations, relative to uninvolved surrounding skin. These studies suggest that the p21-ras protein controls signal transduction pathways capable of modulating AHR function.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 CYP1A1/metabolism , Genes, ras , Receptors, Aryl Hydrocarbon/metabolism , Animals , Blotting, Western , Breast/metabolism , Cell Line , Cytochrome P-450 CYP1B1 , Cytochrome P-450 Enzyme System/metabolism , Down-Regulation , Humans , Keratinocytes/metabolism , Mice , Mice, Transgenic , Polychlorinated Dibenzodioxins/pharmacology , Quinone Reductases/metabolism , Skin Neoplasms/enzymology , TransfectionABSTRACT
Both large, acute doses of erythropoietin (EPO) and short-term hypoxia increase platelet counts in mice, but long-term hypoxia causes thrombocytopenia. Therefore, we tested the hypothesis that EPO injected in large, chronic doses (a total of 80 U of EPO over a 7-day period) might cause thrombocytopenia. EPO caused increased red blood cell (RBC) production, ie, increased hematocrits, RBC counts, mean cell volume (MCV), and reticulocyte counts (from P less than .05 to P less than .0005), and decreased thrombocytopoiesis, ie, decreased platelet counts, percent 35S incorporation into platelets, and total circulating platelet counts (TCPC) (P less than .0005). Femoral marrow megakaryocyte size was unchanged, but megakaryocyte number was significantly (P less than .005) reduced in mice treated with EPO. EPO-injected mice had increased spleen volumes (P less than .0005), but blood volumes (BV) were unchanged. In EPO-treated, splenectomized mice, RBC production was also increased (P less than .05 to P less than .0005) and platelet counts, TCPC, and percent 35S incorporation into platelets were decreased (P less than .05), but BV was not altered. Therefore, the decrease in platelet counts observed in EPO-treated mice was not due to increased BV or to an enlarged spleen. In other experiments, mice were rendered acutely thrombocytopenic to increase thrombocytopoiesis, and platelet and RBC production rates were determined. In mice with elevated thrombocytopoiesis, RBC counts, hematocrits, percent 59Fe RBC incorporation values, and MCV were decreased (P less than .05 to P less than .0005). Because 59Fe RBC incorporation and MCV were not elevated, the decrease in RBC counts and hematocrits does not appear to be due to bleeding. Therefore, we show that large, chronic doses of EPO increase erythropoiesis and decrease thrombocytopoiesis. Conversely, acute thrombocytopenia causes increased thrombocytopoiesis and decreased erythropoiesis. These findings support the hypothesis of competition between precursor cells of the erythrocytic and megakaryocytic cell lines (stem-cell competition) as the cause of thrombocytopenia in EPO-treated mice and the cause of anemia in mice whose platelet production rates were increased.
Subject(s)
Blood Platelets/metabolism , Erythropoietin/toxicity , Thrombocytopenia/chemically induced , Animals , Blood Platelets/drug effects , Blood Platelets/immunology , Blood Volume/drug effects , Bone Marrow/pathology , Erythrocyte Count , Hematocrit , Immune Sera/administration & dosage , Male , Megakaryocytes/drug effects , Megakaryocytes/pathology , Mice , Mice, Inbred C3H , Platelet Count/drug effects , Recombinant Proteins/toxicity , Splenectomy , Sulfates/blood , Sulfur Radioisotopes , Thrombocytopenia/blood , Thrombocytopenia/pathologyABSTRACT
In an effort to explain the different platelet production capabilities of male and female mice, megakaryocyte and platelet indices were measured on castrated male and oophorectomized female C3H and BALB/c mice, along with suitable intact controls. In agreement with our previous work, intact male BALB/c mice had higher platelet counts and percent incorporation of sulfur 35 into platelet values than did intact female BALB/c mice. Also, both intact BALB/c and C3H male mice had higher platelet counts than their castrated counterparts. Fewer femoral megakaryocytes were found in intact BALB/c and C3H male mice than in their female counterparts (p less than 0.05), but only BALB/c male mice had larger megakaryocytes than BALB/c female mice (p less than 0.0005). Castration caused increased numbers and decreased sizes of megakaryocytes (p less than 0.05) in both strains of mice, but oophorectomy did not change the characteristics of megakaryocytes in these mice. In all treatment groups, C3H mice had megakaryocytes with higher average deoxyribonucleic acid content than did BALB/c mice (p less than 0.0005), that is, BALB/c mice had greater percentages of 8N and 16N megakaryocytes than did C3H mice, but C3H mice had higher proportions of 32N and 64N megakaryocytes than did BALB/c mice (p less than 0.05 to p less than 0.0005). Although a difference in megakaryocyte ploidy was not detected between intact male and intact female C3H mice, BALB/c female mice had elevated percentages of low ploidy classes (8N) when compared with BALB/c male mice (p less than 0.005). Intact male C3H mice had higher percentages of 16N megakaryocytes (p less than 0.05) than did their neutered counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Blood Platelets/cytology , Hematopoiesis , Megakaryocytes/cytology , Mice, Inbred BALB C/blood , Mice, Inbred C3H/blood , Sex Characteristics , Animals , Blood Cell Count , Blood Platelets/chemistry , Blood Platelets/metabolism , Cell Differentiation , Cell Division , DNA/analysis , DNA/metabolism , Female , Flow Cytometry , Male , Megakaryocytes/chemistry , Megakaryocytes/metabolism , Mice , Orchiectomy , Ovariectomy , Ploidies , Species Specificity , Sulfur/metabolism , Sulfur RadioisotopesABSTRACT
In an effort to explain the different platelet production capabilities of both normal and hypoxic male and female C3H and BALB/c mice, megakaryocyte size and number were determined utilizing bone marrow from both normal and hypoxic mice. The results indicate that normal BALB/c female mice have increased numbers of megakaryocytes, but of smaller size compared with either BALB/c male mice or to both sexes of C3H mice. An inverse relationship between the size and number of megakaryocytes was found in both normal and hypoxic mice; therefore, to evaluate total megakaryocyte characteristics, we calculated total megakaryocyte masses (TMM). With hypoxia, megakaryocyte number decreased, whereas megakaryocyte size increased. Despite the increase in megakaryocyte size, hypoxia caused a significant decrease in TMM (P less than 0.005) in all mice, but female C3H mice had higher TMM (P less than 0.05) than did female BALB/c mice. These data show that hypoxia decreases TMM in mice, and that the effect is greater in C3H mice than in BALB/c mice.
Subject(s)
Hypoxia/pathology , Megakaryocytes/pathology , Animals , Cell Count , Female , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Sex Factors , Species SpecificityABSTRACT
Thrombocytopenia develops with prolonged exposure to hypoxia. Although decreases in megakaryocyte numbers due to hypoxia have been well documented, the effects of hypoxia on megakaryocyte DNA content have not been reported. In this study, megakaryocytopoiesis and platelet production were compared in both C3H mice (whose megakaryocyte modal ploidy class is 32N) and C57/BL mice (whose modal ploidy class is 16N), by enclosure in cages covered with silicone-rubber membranes. After equilibration, O2 levels inside the cages were 6%-7%. Hematocrits, platelet counts, platelet sizes, percent 35S incorporation into platelets, megakaryocyte size and number, and megakaryocyte DNA content of mice were measured before and at various days after hypoxia. Although hematocritis increased and platelet counts decreased in both strains of mice with time in hypoxic chambers, megakaryocyte and platelet responses of C3H mice differed from those of C57/BL mice in several respects; hematocrits of C3H mice were higher and platelet counts were lower than those in C57/BL mice. C3H mice produced larger platelets than C57/BL mice in response to hypoxia. Total circulating platelet counts (TCPC) and total circulating platelet masses (TCPM) of both mouse strains showed similar biphasic responses, that is, elevated TCPC and TCPM on days 2-4 and decreased values after 6-14 days of hypoxia. However, hypoxic C3H mice had lower TCPC on days 4-14 and lower TCPM on days 10-14 of hypoxia than C57/BL mice. Both C3H and C57/BL mice had decreased megakaryocyte numbers at 6-10 days of hypoxia, but only C3H mice had decreased numbers of megakaryocytes at day 14. Elevated megakaryocyte size was observed in both mouse strains at day 14 of hypoxia. However, after hypoxia, C3H mice showed a greater depression in megakaryocyte number and a larger increase in megakaryocyte sizes than did C57/BL mice. C3H mice maintained 32N as the modal megakaryocyte DNA content through day 10 of hypoxia, but 64N was the modal megakaryocyte DNA content at day 14; 16N remained the modal megakaryocyte DNA content in hypoxic C57/BL mice. Hypoxic C3H mice had an increase in 16N megakaryocytes after 6 days of hypoxia, followed by an increase in the proportion of 64N cells at 14 days compared to values of untreated C3H control mice. Hypoxic C57/BL mice had an increased proportion of 16N cells at 6 days but a decreased proportion of 32N cells at 14 days. These studies demonstrate that the decreased platelet production resulting from prolonged exposure to hypoxia is primarily the result of decreased differentiation of hematopoietic precursors into the megakaryocyte lineage rather than decreased megakaryocyte DNA content, because higher ploidy classes actually increase as thrombocytopenia becomes more severe. Stem cell competition could explain the findings of reduced platelet production and increased red blood cell production in both strains of mice after exposure to hypoxia.
Subject(s)
Blood Platelets/cytology , DNA/analysis , Hypoxia/physiopathology , Megakaryocytes/chemistry , Ploidies , Animals , Bone Marrow/metabolism , Cell Differentiation/physiology , Cell Division/physiology , DNA/genetics , Hematocrit , Hematopoiesis/physiology , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Oxygen/pharmacology , Platelet Count , Sulfur RadioisotopesABSTRACT
Recent work revealed that mice in which platelet function was inhibited by acetylsalicylic acid (ASA) treatment showed evidence of increased platelet production. It was proposed that poorly functioning platelets gave rise to elevated thrombocytopoiesis by causing the release and action of thrombopoietin. However, direct evidence is lacking. Therefore, in the work reported here, plasma from mice treated with ASA was injected into normal recipient mice in an attempt to document the existence of the humoral factor. Compared with control mice given normal plasma, the injection of mice with plasma from ASA-treated mice resulted in increased thrombocytopoiesis, as evidenced by significant increases in the percentage of 35S incorporation into platelets, larger platelet size, and elevated megakaryocyte precursor cells (the small acetylcholinesterase-positive cell). For a positive control, additional mice were treated with plasma from animals made thrombocytopenic by an injection of antiplatelet serum. These mice also showed significant increases in thrombocytopoiesis. The results support the hypothesis that platelet production in ASA-treated mice is elevated by release and action of thrombopoietin.
Subject(s)
Aspirin/pharmacology , Thrombopoietin/biosynthesis , Animals , Blood Platelets/cytology , Blood Platelets/metabolism , Cell Division/drug effects , Male , Mice , Mice, Inbred C3H , Platelet Activation/drug effects , Platelet Activation/physiologyABSTRACT
A thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) derived from human embryonic kidney (HEK) cells is known to increase platelet production and to increase the number of morphologically unrecognizable early megakaryocytes, ie, small acetylcholinesterase-positive (SAChE+) cells in mice. Other recent studies have concluded that interleukin-6 (IL-6) also stimulates murine megakaryocytopoiesis both in vitro and in vivo. Some workers have suggested that IL-6 is thrombopoietin. Therefore, the purpose of this study was to compare the effects of TSF and IL-6 on percent 35S incorporation into platelets, platelet sizes, and the percentages of SAChE+ cells in C3H mice, and to determine if they produce the same or different responses. The results showed that two or four injections of a partially purified TSF (total dose of 2 or 4 units (U) over a 1- or 2-day period) increased percent 35S incorporation into platelets (P less than .005) and platelet sizes (P less than .005) of both normal and rebound-thrombocytotic mice when compared with values from other mice treated with human serum albumin, the carrier protein for both TSF and IL-6. In eight separate experiments, it was shown that IL-6 (40,000 U, 4 micrograms), when given to rebound-thrombocytotic mice in four injections over a 2-day period, produced a small but significant (P less than .005) increase in percent 35S incorporation into platelets. Additional studies showed that negative results were obtained when similar high doses of IL-6 were administered in two doses over a 1-day period. TSF, but not IL-6, stimulated an increase in platelet sizes of normal mice (P less than .005 to 0.0005); however, IL-6 increased platelet sizes of rebound-thrombocytotic mice when given in two of four injections (P less than .05 to .0005). Also, IL-6, but not TSF, caused anemia in normal mice (P less than .0005) that were given two injections and tested 3 days later. TSF stimulated an increase (P less than .005) in the percentage of SA-ChE+ cells; whereas IL-6, even at high doses, did not. Because of the observed differences in biologic responses of these two cytokines, we conclude that TSF and IL-6 are separate entities.
Subject(s)
Blood Platelets/cytology , Hematopoiesis , Interleukin-6/pharmacology , Megakaryocytes/cytology , Thrombopoietin/pharmacology , Acetylcholinesterase/metabolism , Animals , Blood Platelets/metabolism , Hematocrit , Male , Megakaryocytes/metabolism , Mice , Mice, Inbred C3H , Sulfur Radioisotopes/metabolismABSTRACT
The present work reports the preparation of a highly bioactive and stable thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) by a four-step purification procedure, i.e., Sephadex column chromatography, ethanol precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and reverse phase-high performance liquid chromatography. The molecular weight (MW) of the purified product depended upon the method of purification, i.e., using denaturing buffers at 56 degrees C for 10 min, the MW was approximately 30,000 daltons; whereas, after preparing in denaturing buffers and heating to 100 degrees C for 10 min, the purified protein had an apparent MW of approximately 15 kd. Both moieties had significant biological activity. The data indicate that TSF may exist normally as a dimer (30 kd), but can disassociate to 15 kd without loss of bioactivity. The present work illustrates that the purified TSF has an isoelectric pH of 4.47 and exists in trace amounts in human embryonic kidney (HEK) cell culture media. The final product prepared in the presence of Tween-20 had a specific activity of approximately 21,000 U of TSF per mg of protein, representing a purification factor of approximately 164,000. Using this four-step purification procedure, a homogeneous product was obtained as judged by SDS-PAGE and chromatofocusing. This purified material will be suitable for further studies, including amino acid sequencing.
Subject(s)
Glycoproteins/isolation & purification , Thrombopoietin/isolation & purification , Chemical Precipitation , Chromatography , Chromatography, High Pressure Liquid , Drug Stability , Electrophoresis, Polyacrylamide Gel , Ethanol , Humans , Molecular WeightABSTRACT
Previously, recombinant erythropoietin (rEpo) was shown to increase the number and size of megakaryocytic colonies in vitro, and in vivo it elevates the number of megakaryocytes in mouse spleens. To test the hypothesis that rEpo would stimulate platelet production in mice, both normal mice and mice in rebound-thrombocytosis were injected with rEpo and the %35S incorporation into platelets was measured. A thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) was used as a positive control. rEpo increased isotopic incorporation into platelets of both normal mice and mice in rebound-thrombocytosis, as did TSF, but required large doses (15 U rEpo/mouse). In other mice, hematocrits, platelet counts, platelet sizes, and 24-hr %35S incorporation into platelets were measured 2 days after injection of two equally divided doses of either rEpo or TSF. Significant increases in both platelet sizes and %35S incorporation into platelets were found after injections of 15 U rEpo/mouse or 2.3 U TSF/mouse. These data indicate that rEpo, at high doses, will stimulate platelet production in mice, and may suggest molecular similarities between rEpo and TSF and their ability to compete for common receptor sites on megakaryocytes and their progenitor cells.