RESUMO
5'-Nucleotidases are the catabolic members of the substrate cycles postulated to be involved in the regulation of intracellular deoxyribonucleoside triphosphate pools. Here, we attempt to identify the nature of the nucleotidases. Earlier, we constructed various mammalian cell lines that can be induced to overproduce the high K(m) 5'-nucleotidase (hkm-NT) or the 5'(3')-deoxynucleotidase (dNT-1). Now we labeled control and induced human 293 cells and hamster V79 cells with radioactive hypoxanthine or uridine and during a chase measured quantitatively the metabolism of ribo- and deoxyribonucleotides, DNA replication, and excretion of nucleosides into the medium. Overproduction of hkm-NT greatly increased excretion of inosine and guanosine but did not affect adenosine or deoxyribonucleosides. dNT-1 overproduction increased excretion of deoxycytidine, thymidine, and in particular deoxyuridine but also uridine and cytidine. We conclude that the hkm-NT is not involved in the regulation of deoxyribonucleotide pools but affects IMP and GTP pools. dNT-1, instead, appears to be the catabolic arm of substrate cycles regulating pyrimidine nucleotide pools.
Assuntos
5'-Nucleotidase/fisiologia , Citosol/enzimologia , Nucleotídeos/metabolismo , Animais , Ciclo Celular , Linhagem Celular , Cricetinae , DNA/biossíntese , Humanos , Nucleosídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismoRESUMO
Human 293 cells were stably transfected with a plasmid introducing a receptor for the ecdysone analog muristerone. The cells were further stably transfected with muristerone-inducible expression vectors carrying either the cDNA for the human high K(M) 5'-nucleotidase or the coding sequence of the nucleotidase linked to the 5'-end of the sequence for the green fluorescent protein. Upon induction, both types of transfectants overproduced nucleotidase activity in a time- and dose-dependent manner. Western blots gave values close to the expected subunit molecular masses of 65 and 92 kDa, respectively, excluding processing of the induced proteins. Cells induced to overexpress the nucleotidase showed a decreased growth rate and contained smaller pools of each of the four common ribonucleoside triphosphates. They showed no increased resistance to the toxicity of 2-chlorodeoxyadenosine.
Assuntos
5'-Nucleotidase/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , 5'-Nucleotidase/análise , 5'-Nucleotidase/metabolismo , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/metabolismo , Antineoplásicos/farmacologia , Western Blotting , Células Cultivadas , Cladribina/farmacologia , Clonagem Molecular , Citidina Trifosfato/análise , Citidina Trifosfato/metabolismo , DNA Complementar , Ecdisterona/análogos & derivados , Ecdisterona/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Proteínas de Fluorescência Verde , Guanosina Trifosfato/análise , Guanosina Trifosfato/metabolismo , Humanos , Indicadores e Reagentes/metabolismo , Rim/citologia , Proteínas Luminescentes/genética , Plasmídeos , Receptores de Esteroides/genética , Transfecção , Uridina Trifosfato/análise , Uridina Trifosfato/metabolismoRESUMO
Hyaluronan (HL), a naturally occurring glycosaminoglycan, has been chemically modified through the esterification of its carboxylic groups with different types of alcohol. The physico-chemical properties of these new biopolymers allow the preparation of many biomaterials which may be used in several medical applications. In the present study both the biocompatibility and biodegradation of some water-insoluble HL esters have been evaluated, either as raw material or as manufactured devices after subcutaneous and intraperitoneal implantation in male rats. The inflammatory response and the degree of resorption for each tested material are reported. The relationships between the degree of esterification and the type of alcohol used with the above parameters are also investigated.
Assuntos
Materiais Biocompatíveis/farmacologia , Ácido Hialurônico/análogos & derivados , Animais , Materiais Biocompatíveis/metabolismo , Biodegradação Ambiental , Tecido Conjuntivo/patologia , Ácido Hialurônico/metabolismo , Ácido Hialurônico/farmacologia , Masculino , Teste de Materiais , Peritônio/patologia , Próteses e Implantes/efeitos adversos , Ratos , Ratos WistarRESUMO
Two in vitro systems (the DNA synthetic response to mycobacterial antigens and cytotoxicity against lymphoid cells) were used to analyse the effect of thymolymphotropin (TLT) on peripheral blood mononuclear cells (PBMC). Purified protein derivative of mycobacteria (PPD)-driven T-cell proliferation in low-responder donors was increased by the combined treatment with TLT and suboptimal doses of recombinant interleukin 2 (IL-2). Similarly, the activities of natural killer (NK) cells and lymphokine-activated killer (LAK) cells have been enhanced in PBMC cultures pretreated with TLT. Also, TLT showed an enhancing effect on the development of LAK cells capable of lysing Epstein-Barr virus (EBV)-transformed B-lymphocytes infected or uninfected with the human immunodeficiency virus (HIV).
Assuntos
Interleucina-2/administração & dosagem , Leucócitos Mononucleares/imunologia , Extratos do Timo/administração & dosagem , Citotoxicidade Imunológica , HIV/fisiologia , Infecções por HIV/imunologia , Infecções por HIV/terapia , Humanos , Imunoterapia , Técnicas In Vitro , Ativação Linfocitária , Linfócitos T/imunologia , Tuberculina/imunologia , Replicação ViralRESUMO
The ability of liposomes containing a new lipophilic muramyl peptide derivative, MDP-L-alanyl-cholesterol (MTP-CHOL), to induce peritoneal macrophage cytostatic activity and alveolar macrophage cytotoxic activity toward tumor cell targets in vitro was determined. MTP-CHOL was shown to be efficiently incorporated and subsequently retained in distearoylphosphatidylcholine/phosphatidylserine liposomes (DSPC/PS; 7:3 molar ratio), whereas hydrosoluble muramyl dipeptide (MDP) was rapidly lost due to leakage. Liposomes containing MTP-CHOL were able to stimulate mouse peritoneal macrophage cytostatic activity under conditions where free MDP was without effect. MTP-CHOL incorporated into liposomes was approximately eightfold more effective than liposomes containing entrapped MDP and 7,400-fold more effective than free MDP in inducing rat alveolar macrophage cytotoxic activity. These results provide evidence that the coupling of MDP to a lipophilic molecule, cholesterol, results in the formation of a viable liposome formulation that is a potent inducer of macrophage-mediated antitumor activity.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Ésteres do Colesterol/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Acetilmuramil-Alanil-Isoglutamina/síntese química , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Animais , Linhagem Celular , Ésteres do Colesterol/síntese química , Relação Dose-Resposta a Droga , Feminino , Lipossomos/administração & dosagem , Sarcoma de Mastócitos/imunologia , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos DBA , Camundongos EndogâmicosRESUMO
The effect of two muramyl dipeptides, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and N-acetylmuramyl-D-alanyl-D-isoglutamine (MDP(D-D)), on the in vitro growth of two murine ascitic plasmacytomas, MOPC 173 and TEPC 15, and on an ascitic lymphoma cell line, ABPL2, was studied. The ability of the muramyl dipeptides to inhibit tumor cell growth was compared with a sonicated antigenic preparation of Mycobacterium vaccae (Vaccin), known to have macrophage stimulation activity. The growth of all three ascitic cell lines was inhibited by both muramyl dipeptides and Vaccin. Macrophage-depletion of the ascitic cell populations led to an increase in cell growth of TEPC 15 and MOPC 173, and a decrease in ABPL2. A reduction or loss of the inhibitory activity of the muramyl dipeptides or Vaccin was also observed, and no inhibitory activity was found when the tumor cell lines were cultured in vitro to render them macrophage-free. The inhibitory activity of MDP or MDP(D-D) was restored when purified ascitic macrophages were added to the in vitro cultured cell lines. It was demonstrated that a minimum number of macrophages were necessary for the expression of inhibitory activity. Indomethacin, a PG-synthetase inhibitor, was found to act in a synergistic manner with MDP and MDP(D-D) in inhibiting TEPC 15, but antagonized the effect of these two agents on ABPL2. The lymphoma cell line ABPL2 appeared to be the most sensitive to inhibition by MDP(D-D), a nonpyrogenic, adjuvant-inactive stereoisomer of MDP.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/farmacologia , Antineoplásicos/farmacologia , Linfócitos B/imunologia , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/imunologia , Mycobacterium/imunologia , Animais , Antígenos de Bactérias/imunologia , Linhagem Celular , DNA de Neoplasias/biossíntese , Linfoma/imunologia , Linfoma/terapia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Plasmocitoma/imunologia , Plasmocitoma/terapiaRESUMO
The ability of a member of a new class of lipophilic muramyl dipeptide (MDP) derivative, muramyl dipeptide-glyceryldipalmitate (MDP-GDP), to induce alveolar macrophage cytotoxic activity in vitro towards B16 melanoma cells when incorporated into two types of liposome was studied. MDP-GDP incorporated into conventionally prepared liposomes formulated from distearoylphosphatidylcholine and phosphatidylserine (7:3 molar ratio) was 10-fold more effective than liposomes containing MDP, and 7000-fold more effective than free MDP in inducing macrophage cytotoxic activity. MDP-GDP incorporated into freeze-dried liposomes was 50,000- to 100,000-fold more effective than free MDP in inducing such activity. Freeze-dried liposomes containing MDP-GDP were efficiently localized in the lungs of normal mice, and induced cytotoxic activity in the alveolar macrophages. Such liposomes were able to significantly reduce the pulmonary metastatic burden of mice carrying the B16 melanoma. These data provide evidence that this class of lipophilic MDP derivative, when incorporated into freeze-dried liposomes, is a potent inducer of macrophage cytotoxic activity in vitro and in situ, and has antitumor activity in vivo. In addition, the use of a freeze-drying procedure allows the preparation and long-term storage of reproducible liposome formulations.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Lipossomos/administração & dosagem , Neoplasias Pulmonares/secundário , Ativação de Macrófagos , Melanoma/terapia , Triglicerídeos/uso terapêutico , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Acetilmuramil-Alanil-Isoglutamina/uso terapêutico , Animais , Citotoxicidade Imunológica/efeitos dos fármacos , Liofilização , Imunoterapia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Macrófagos/efeitos dos fármacos , Masculino , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Endogâmicos F344 , Triglicerídeos/farmacologiaRESUMO
The immunological activity of Momordica Charantia inhibitor (MCI) and of Pokeweed antiviral protein (PAP-S), 30,000 daltons plant proteins possessing close similarity to Ricin A chain as inhibitor of protein synthesis, was investigated in mice. In vivo, single nontoxic injections of microgram amount of these substances delayed H2-incompatible skin allograft rejection, splenocyte responsiveness to ConA and PHA, but not to LPS, and abrogated the PFC response to a T-dependent (SRBC) antigen while totally sparing that to a T-independent (S III) stimulus. Injection of these substances could also reduce NK cell activity while increasing macrophage-mediated spontaneous cytotoxicity. In vitro, MCI and PAP-S at non-cytotoxic concentrations inhibited lymphoid cell responsiveness to PHA and ConA, but not to LPS, and markedly enhanced macrophage-dependent cytotoxicity.
Assuntos
N-Glicosil Hidrolases , Proteínas de Plantas/imunologia , Animais , Formação de Anticorpos/efeitos dos fármacos , Rejeição de Enxerto/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Inativadoras de Ribossomos Tipo 1 , Proteínas Inativadoras de Ribossomos Tipo 2 , Baço/imunologiaRESUMO
In vitro preculture of C3H/He splenocytes for 48 to 96 hr induces aspecific suppressor cells evaluated by their ability to reduce 3H-TdR uptake by fresh syngeneic splenocytes stimulated by different amounts of Concanavalin A (Con A) in vitro. This suppressive activity is obtained in medium containing 10% heat-inactivated fetal calf serum (FCS) or similar amounts of heat-inactivated bovine serum, syngeneic, or allogeneic murine sera but not by unheated FCS. Suppressive activity is resistant to mitomycin C and radiation up to 5,000 R. Exposure of precultured cells to carbonyl iron or plastic adherence, but not to anti-Thy 1.2 serum plus complement, results in removal of the suppressive activity.
Assuntos
Terapia de Imunossupressão , Linfócitos/imunologia , Animais , Células Cultivadas , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Baço/citologia , Baço/efeitos da radiaçãoRESUMO
The presence of suppressor cells in the spleens of C57BL/6 mice bearing Lewis lung carcinoma was investigated with the use of the in vitro lymphoproliferative response to mitogens and the graft-versus-host reaction (GVHR) as test systems. Splenocytes from tumor-bearing mice showed a lower response to mitogens when obtained 15-27 days after tumor transplant. In parallel, these cells were capable of suppressing the response of normal spleen cells to mitogens and their capacity to mount a GVHR in allogeneic hosts. Treatments with procedures known to remove adherent phagocytes, but not treatments with anti-Thy 1.2 serum plus complement, removed the suppressive activity observed.