RESUMO
Prosthetic joint infection (PJI) is a well-known and persisting problem. Active release coatings have promise to provide early protection to an implant by eradicating small colony biofilm contaminants or planktonic bacteria that can form biofilm. Traditional antibiotics can be limited as active release agents in that they have limited effect against biofilms and develop resistance at sub-lethal concentrations. A unique first-in-class compound (CZ-01127) was assessed as the active release agent in a silicone (Si)-based coating to prevent PJI in a sheep model of joint space infection. Titanium (Ti) plugs contained a porous coated Ti (PCTi) region and polymer-coated region. Plugs were implanted into a femoral condyle of sheep to assess the effect of the Si polymer on cancellous bone ingrowth, the effect of CZ-01127 on bone ingrowth, and the ability of CZ-01127 to prevent PJI. Microbiological results showed that CZ-01127 was able to eradicate bacteria in the local region of the implanted plugs. Data further showed that Si did not adversely affect bone ingrowth. However, bacteria that reached the joint space (synovium) were not fully eradicated. Outcomes suggested that the CZ-01127 coating provided local protection to the implant system in a challenging model, the design of which could be beneficial for testing future antimicrobial therapies for PJI. STATEMENT OF SIGNIFICANCE: Periprosthetic joint infection (PJI) is now commonplace, and constitutes an underlying problem that patients and physicians face. Active release antibiotic coatings have potential to prevent these infections. Traditional antibiotics are limited in their ability to eradicate bacteria that reside in biofilms, and are more susceptible to resistance development. This study addressed these limitations by testing the efficacy of a unique antimicrobial compound in a coating that was tested in a challenging sheep model of PJI. The unique coating was able to eradicate bacteria and prevent infection in the environment adjacent to the implant. Bacteria that escaped into the joint space still caused infection, yet benchmark data can be used to optimize the coating and translate it toward clinical use.
Assuntos
Antibacterianos , Materiais Revestidos Biocompatíveis , Infecções Relacionadas à Prótese , Espermidina/análogos & derivados , Animais , Antibacterianos/química , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacocinética , Materiais Revestidos Biocompatíveis/farmacologia , Modelos Animais de Doenças , Implantes de Medicamento/química , Implantes de Medicamento/farmacocinética , Implantes de Medicamento/farmacologia , Feminino , Porosidade , Infecções Relacionadas à Prótese/tratamento farmacológico , Infecções Relacionadas à Prótese/microbiologia , Infecções Relacionadas à Prótese/patologia , Ovinos , Espermidina/química , Espermidina/farmacocinética , Espermidina/farmacologiaAssuntos
Farmacorresistência Fúngica/genética , Genes Essenciais , Poliaminas/farmacologia , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica , Leucina/farmacocinética , Mutação , Saccharomyces cerevisiae/genética , Espermidina/farmacocinética , Espermidina/farmacologia , Espermina/farmacologia , Fatores de Transcrição/genéticaRESUMO
Molecular umbrella provided a promising avenue for the design of the intracellular delivery of hydrophilic therapeutic agents. However, the limited understanding of its cellular uptake would be a roadblock to its effective application. Herein, we investigate the ability and mechanism of cellular entry of a fluorescently labeled diwalled molecular umbrella, which was synthesized from cholic acid, spermine, and 5-carboxyfluorescein, into Hela cells, with the extent of uptake analyzed by confocal fluorescence microscopy and flow cytometry. It is found that the as-synthesized diwalled molecular umbrella can greatly facilitate cellular uptake of hydrophilic agent, 5-carboxyfluorescein. In vitro experiments with diffuse marker, endocytic marker, and inhibitors suggested that several distinct uptake pathways (e.g., passive diffuse, clathrin-mediated endocytosis, and caveolae/lipid-raft-dependent endocytosis) are involved in the internalization of diwalled molecular umbrella. These results, together with its low toxicity and good biocompatibility, thus demonstrate the suitability of molecular umbrella for application as vectors in drug delivery systems.
Assuntos
Materiais Biocompatíveis/metabolismo , Membrana Celular/metabolismo , Ácido Cólico/metabolismo , Portadores de Fármacos/metabolismo , Corantes Fluorescentes/metabolismo , Espermidina/metabolismo , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacocinética , Transporte Biológico , Membrana Celular/química , Células Cultivadas , Ácido Cólico/química , Ácido Cólico/farmacocinética , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Sistemas de Liberação de Medicamentos , Fluoresceínas/química , Fluoresceínas/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Conformação Molecular , Espermidina/química , Espermidina/farmacocinética , Relação Estrutura-AtividadeRESUMO
The utility of polyamines as vectors for the intracellular transport of iron chelators is further described. Consistent with earlier results with polyamine analogues, these studies underscore the importance of charge in the design of polyamine-vectored chelators. Four polyamine conjugates are synthesized, two of terephthalic acid [N(1)-(4-carboxy)benzoylspermine (7) and its methyl ester (6)] and two of (S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid [(S)-4'-(HO)-DADFT] [(S)-4,5-dihydro-2-[2-hydroxy-4-(12-amino-5,9-diazadodecyl-oxy)phenyl]-4-methyl-4-thiazolecarboxylic acid (10) and its ethyl ester (9)]. These four molecules were evaluated in murine leukemia L1210 cells for their impact on cell proliferation (48- and 96-h IC(50) values), their ability to compete with spermidine for the polyamine transport apparatus (K(i)), and their intracellular accumulation. The data revealed that when neutral molecules (cargo fragments) were fixed to the polyamine vector, the conjugates competed well with spermidine for transport and were accumulated intracellularly to millimolar levels. However, this was not the case when the cargo fragments were negatively charged. Metabolic studies of the polyamine-vectored (S)-4'-(HO)-DADFTs in rodents indicated that not only did the expected deaminopropylation step occur, but also a surprisingly high level of oxidative deamination at the terminal primary nitrogens took place. Finally, the iron-clearing efficiency of the (S)-4'-(HO)-DADFT conjugates was determined in a bile-duct-cannulated rodent model. Attaching the ligand to a polyamine vector had a profound effect on increasing the iron-clearing efficiency of this chelator relative to its parent drug.
Assuntos
Quelantes de Ferro/administração & dosagem , Quelantes de Ferro/química , Poliaminas/química , Espermina/análogos & derivados , Espermina/administração & dosagem , Espermina/química , Tiazóis/administração & dosagem , Tiazóis/química , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Eletricidade , Ésteres/administração & dosagem , Ésteres/química , Ésteres/farmacocinética , Quelantes de Ferro/farmacocinética , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Espermidina/química , Espermidina/farmacocinética , Espermina/farmacocinética , Relação Estrutura-Atividade , Tiazóis/farmacocinéticaRESUMO
In the course of our investigations aimed at improving the biological characteristics of iodobenzamides for melanoma therapeutic applications, four new derivatives containing a spermidine chain have been prepared and radiolabeled with (125)I. In vitro studies showed that all compounds displayed high affinity for melanin superior to the reference compound BZA, thus validating our experimental approach. In vivo biodistribution was investigated in B16 melanoma-bearing mice. All four compounds, particularly benzamide 3, showed accumulation in the tumor, but lower, however, than that of BZA. Moreover, high concentrations of radioactivity in other organs, namely, the liver and lung, demonstrated nonspecific tumoral uptake. In view of these results, compounds 1 2 3 4 do not appear to be suitable radiopharmaceuticals for melanoma radionuclide therapy.
Assuntos
Benzamidas/farmacocinética , Biomarcadores Tumorais/metabolismo , Radioisótopos do Iodo/farmacocinética , Melaninas/metabolismo , Melanoma/metabolismo , Espermidina/farmacocinética , Animais , Benzamidas/uso terapêutico , Carga Corporal (Radioterapia) , Linhagem Celular Tumoral , Radioisótopos do Iodo/uso terapêutico , Masculino , Melanoma/radioterapia , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Ligação Proteica , Doses de Radiação , Radiometria/métodos , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/uso terapêutico , Espermidina/uso terapêutico , Distribuição TecidualRESUMO
It has been reported that Gap1p on the plasma membrane of Saccharomyces cerevisiae can catalyze the uptake of many kinds of amino acids. In the present study, we found that Gap1p also catalyzed the uptake of putrescine and spermidine, but not spermine. The Km and Vmax values for putrescine and spermidine were 390 and 21 microM, and 4.6 and 0.59 nmol/min/mg protein, respectively. The uptake of putrescine was strongly inhibited by basic amino acids, lysine, arginine, and histidine, whose Ki values were 25-35 microM. Thus, it is deduced that spermidine and basic amino acids have almost the same affinity for Gap1p. When the concentrations of amino acids in the medium were reduced to one-third and 0.5 mM putrescine or 0.1 mM spermidine was added to the medium, accumulation of putrescine or spermidine by Gap1p was observed. Furthermore, when yeast was transformed with the GAP1 gene and cultured in the presence of 60 mM putrescine, cell growth was inhibited through overaccumulation of putrescine. GAP1 mRNA was found to be induced by polyamines. This is the first report of the identification, at a molecular level, of a polyamine uptake protein on the plasma membrane in eukaryotes.
Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Membrana Celular/metabolismo , Putrescina/farmacocinética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Espermidina/farmacocinética , Membrana Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Proteínas de Membrana/metabolismo , Taxa de Depuração Metabólica , Putrescina/administração & dosagem , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Espermidina/administração & dosagem , Espermina/administração & dosagem , Espermina/farmacocinéticaRESUMO
Metabolically stable polyamine derivatives may serve as useful surrogates for the natural polyamines in studies aimed to elucidate the functions of individual polyamines. Here we studied the metabolic stability of alpha-methylspermidine, alpha-methylspermine, and bis-alpha-methylspermine, which all have been reported to fulfill many of the putative physiological functions of the natural polyamines. In vivo studies were performed with the transgenic rats overexpressing spermidine/spermine N(1)-acetyltransferase. alpha-Methylspermidine effectively accumulated in the liver and did not appear to undergo any further metabolism. On the other hand, alpha-methylspermine was readily converted to alpha-methylspermidine and spermidine; similarly, bis-alpha-methylspermine was converted to alpha-methylspermidine to some extent, both conversions being inhibited by the polyamine oxidase inhibitor N(1), N(2)-bis(2,3-butadienyl)-1,4-butanediamine. Furthermore, we used recombinant polyamine oxidase, spermidine/spermine N(1)-acetyltransferase, and the recently discovered spermine oxidase in the kinetic studies. In vitro studies confirmed that methylation did not protect spermine analogs from degradation, whereas the spermidine analog was stable. Both alpha-methylspermidine and bis-alpha-methylspermine overcame the proliferative block of early liver regeneration in transgenic rats and reversed the cytostasis induced by an inhibition of ornithine decarboxylase in cultured fetal fibroblasts.
Assuntos
Poliaminas/síntese química , Poliaminas/farmacocinética , Espermidina/análogos & derivados , Acetiltransferases/metabolismo , Animais , Animais Geneticamente Modificados , Biotransformação , Estabilidade de Medicamentos , Fibroblastos/citologia , Fibroblastos/metabolismo , Cinética , Fígado/metabolismo , Metilação , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Ratos , Espermidina/síntese química , Espermidina/farmacocinética , Distribuição Tecidual , Poliamina OxidaseRESUMO
The product of the UGA4 gene in Saccharomyces cerevisiae, which catalyzes the transport of 4-aminobutyric acid (GABA), also catalyzed the transport of putrescine. The Km values for GABA and putrescine were 0.11 and 0.69 mM, respectively. The UGA4 protein was located on the vacuolar membrane as determined by the effects of bafilomycin A1 and by indirect immunofluorescence microscopy. Uptake of both GABA and putrescine was inhibited by spermidine and spermine, although these polyamines are not substrates of UGA4. The UGA4 mRNA was induced by exposure to GABA, but not putrescine over 12h. The growth of an ornithine decarboxylase-deficient strain was enhanced by putrescine, and both putrescine and spermidine contents increased, when the cells were expressing UGA4. The results suggest that a substantial conversion of putrescine to spermidine occurs in the cytoplasm even though UGA4 transporter exists on vacuolar membranes.
Assuntos
Membranas Intracelulares/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Putrescina/farmacocinética , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Ácido gama-Aminobutírico/farmacocinética , Azidas/farmacologia , Transporte Biológico/genética , Proteínas de Transporte/metabolismo , Divisão Celular/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de GABA , Macrolídeos/farmacologia , Níquel/química , Níquel/metabolismo , Transportadores de Ânions Orgânicos/genética , Ornitina Descarboxilase/deficiência , Poliaminas/metabolismo , Putrescina/farmacologia , RNA Mensageiro/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae , Espermidina/farmacocinética , Espermina/farmacocinética , Frações Subcelulares/metabolismo , Ácido gama-Aminobutírico/farmacologiaRESUMO
[3H]Spermidine (SPD) was accumulated in subcellular fractions enriched of the nucleus in a temperature-dependent manner with a saturable profile in murine brain. The accumulation reached a plateau within 30 min at 2 degrees C and 30 degrees C, while excess unlabeled SPD significantly inhibited the accumulation at 2 degrees C without markedly affecting that at 30 degrees C when added after equilibrium. The temperature-dependent accumulation of [3H]SPD was significantly inhibited by the triamine SPD and the tetraamine spermine, but not by the diamine putrescine. Phospholipases were invariably effective in significantly inhibiting the accumulation at 30 degrees C in a concentration-dependent manner. Amongst different discrete murine central structures examined, the temperature-dependent [3H]SPD accumulation was highest in neocortex with progressively lower activities in striatum, hypothalamus, spinal cord, medulla-pons, hippocampus, midbrain and cerebellum. These results suggest the possible presence of a hitherto unidentified nuclear transport system for particular polyamines in murine brain.
Assuntos
Temperatura Corporal/fisiologia , Encéfalo/metabolismo , Matriz Nuclear/metabolismo , Espermidina/farmacocinética , Trifosfato de Adenosina , Animais , Transporte Biológico , Encéfalo/citologia , Glicosídeo Hidrolases/farmacologia , Guanosina Trifosfato , Histonas/metabolismo , Immunoblotting , Cinética , Masculino , Camundongos , Fosfolipases/farmacologia , Poliaminas/farmacocinética , Propídio/farmacocinética , Soroalbumina Bovina/farmacologia , Frações Subcelulares/metabolismo , Fatores de Tempo , Trítio/farmacocinética , Aglutininas do Germe de Trigo/farmacologiaRESUMO
Our study demonstrates the capacity of FR160, a catechol iron chelator, to reach and accumulate into infected Plasmodium falciparum erythrocytes and parasites (cellular accumulation ratio between 12 and 43). Steady-state FR160 accumulation is obtained after 2 hr of exposure. After 2 hr exposure, it reaches intracellular levels that are 4- to 10-fold higher in infected red blood cells than those attained in normal erythrocytes. There is quite a good correlation between the accumulation of chloroquine and FR160 in the different strains (r=0.939) and in the IC(50) values (r=0.719). In contrast, the accumulation of FR160 and its activity is poorly correlated (r=0.500), suggesting that activity of FR160 may be independent of its penetration into infected erythrocytes. The mechanism of accumulation is yet unknown but based on inhibitor studies, the uptake of FR160 seems to be not associated with the calcium pump or channel, the potassium channel or the Na(+)/H(+) exchanger. Combinations of FR160 with verapamil, diltiazem, clotrimazole, amiloride, diazoxide, 4-aminopyridine, and picrotoxin should be avoided (antagonistic effects). The potent in vitro activity of FR160 on chloroquine-resistant strains or isolates, its lower toxicity against Vero cells, its mechanisms of action, its capacity to reach rapidly and accumulate into infected erythrocytes suggest that FR160 holds much promise as a new structural lead and effective antimalarial agent or at least a promising adjuvant in treatment of malaria.
Assuntos
Cloroquina/sangue , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidade , Espermidina/análogos & derivados , Espermidina/sangue , Animais , Transporte Biológico , Cloroquina/farmacocinética , Humanos , Ferro/metabolismo , Cinética , Sideróforos/sangue , Sideróforos/farmacocinética , Espermidina/farmacocinéticaRESUMO
BACKGROUND AND AIM: Not only biosynthesis, but also uptake from the intestinal lumen, are important polyamine sources. However, there has been no information regarding dynamic polyamine transport in the small intestine. We evaluated polyamine uptake from the small intestine using a rat ex vivo model. METHODS: The organ block consisting of the small intestine and blood vessels was used. The isolated small intestine was placed in a warmed saline bath and perfused in a non-circulating manner via the superior mesenteric artery. Radio-labeled putrescine, spermidine or spermine (7.4 x 104 Bq), with 1.0 mL of phosphate buffer saline (pH 7.4) was instilled into the jejunal lumen for 1 min. Blood samples from the portal vein were collected and sample radioactivity was determined. In another experiment, an immunohistochemical study of polyamine was performed. RESULTS: After 14C-polyamine instillation, radioactivity in the portal vein samples immediately increased and then decreased gradually. The absorptive pattern did not differ among the three polyamines. The recovery rates from radioactivity at the portal vein among the three polyamines were approximately 61-76% during the initial 10 min after the administration of 14C-polyamine, and were not different from each other. Aminoguanidine, which inhibits putrescine degradation, significantly suppressed initial putrescine uptake and recovery percentage. The intraluminal administration of spermine caused an increase in the immunoreactivity of the spermine antibody in the intestinal villi. CONCLUSION: Luminal polyamines were rapidly absorbed by the intestinal mucosa and then subsequently transferred into the portal vein using a rat ex vivo model. The prior administration of aminoguanidine significantly inhibited initial putrescine transport into the portal vein.
Assuntos
Absorção Intestinal/fisiologia , Poliaminas/farmacocinética , Animais , Transporte Biológico/fisiologia , Mucosa Intestinal/patologia , Mucosa Intestinal/fisiologia , Intestino Delgado/patologia , Intestino Delgado/fisiologia , Masculino , Poliaminas/administração & dosagem , Putrescina/administração & dosagem , Putrescina/farmacocinética , Ratos , Ratos Wistar , Espermidina/administração & dosagem , Espermidina/farmacocinética , Espermina/administração & dosagem , Espermina/farmacocinéticaRESUMO
In Saccharomyces cerevisiae four transporters, Tpo1p-Tpo4p, all members of the major facilitator superfamily, have been shown to confer resistance to polyamines. It was suggested that they act by pumping their respective substrate into the lumen of the vacuole depending on the proton gradient generated by the V-ATPase. Using sucrose gradient ultracentrifugation we found that an hemagglutinin (HA)-tagged Tpo1p as well as its HA-tagged Tpo2p-4p homologues co-localize with plasma membrane markers. Because the HA-tagged Tpo1p carrier protein proved to be functional in conferring resistance to polyamines in TPO1 knockouts, a function of Tpo1p in transport of polyamines across the plasma membrane seemed to be likely. The polyamine transport activity of wild type cells was compared with the respective activity of a TPO1 knockout strain. The results obtained strongly suggest that Tpo1p is a plasma membrane-bound exporter, involved in the detoxification of excess spermidine in yeast. When studying polyamine transport of wild type cells, we furthermore found that S. cerevisiae is excreting putrescine during the fermentative growth phase.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Poliaminas/farmacocinética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Antiporters , Transporte Biológico , Proteínas de Transporte/análise , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Primers do DNA , Resistência a Múltiplos Medicamentos , Cinética , Proteínas de Membrana Transportadoras/análise , Proteínas de Membrana Transportadoras/genética , Proteínas de Transporte de Cátions Orgânicos , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/genética , Espermidina/farmacocinética , Vacúolos/metabolismo , Vacúolos/ultraestruturaRESUMO
A new cationic polymer (PPA-SP), polyphosphoramidate bearing spermidine side chain, was prepared as a non-viral vector for gene delivery. PPA-SP was synthesized from poly(1,2-propylene H-phosphonate) by the Atherton-Todd reaction. The weight average molecular weight of PPA-SP was 3.44x10(4) with a number average degree of polymerization of 90, as determined by GPC/LS/RI method. The average net positive charge per polymer chain was 102. PPA-SP was able to condense plasmid DNA efficiently and formed complexes at an N/P ratio (free amino groups in polymer to phosphate groups in DNA) of 2 and above, as determined by agarose gel electrophoresis. This new gene carrier offered significant protection to DNA against nuclease degradation at N/P ratios above 2, and showed lower cytotoxicity than PLL and PEI in cell culture. The LD(50) of PPA-SP was 85 microg/ml in COS-7 cells, in contrast to 20 and 42 microg/ml for PLL and PEI, respectively. The complexes prepared in saline at N/P ratios of 5 approximately 10 had an average size of 250 nm and zeta-potential of 26 mV. PPA-SP mediated efficient gene transfection in a number of cell lines, and the transfection protocol was optimized in HEK293 cells using a luciferase plasmid as a marker gene. Gene expression mediated by PPA-SP was greatly enhanced when chloroquine was used in conjunction at a concentration of 100 microM. Under the optimized condition, PPA-SP/DNA complexes yield a luciferase expression level closed to PEI/DNA complexes or Transfast mediated transfection. In a non-invasive CNS gene delivery model, PPA-SP/DNA complexes yielded comparable bcl-2 expression as PEI/DNA complexes in mouse brain stem following injection of the complexes in the tongue.
Assuntos
Portadores de Fármacos/administração & dosagem , Terapia Genética/métodos , Polímeros/administração & dosagem , Espermidina/administração & dosagem , Animais , Células COS/efeitos dos fármacos , Células COS/metabolismo , Chlorocebus aethiops , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Polímeros/química , Polímeros/farmacocinética , Espermidina/química , Espermidina/farmacocinética , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
Transport of a polyamine (PA), spermidine (SPMD) into rat brain at various early postischemic periods was studied. Rats underwent 20 min of four-vessel occlusion (4VO) followed by 5, 10, 30 and 60 min of recirculation (RC) periods with natural brain temperature. 3H-aminoisobutyricacid (AIB) and 14C-SPMD were utilised to search dual functions of the blood-brain barrier (BBB); barrier and carrier functions, respectively. Unidirectional blood-to-brain transfer constant (Kin) was calculated for AIB and SPMD in four brain regions-parieto-temporal cortex, striatum, hippocampus and cerebellum. Kin for SPMD ranged between 1.2+/-0.3 x 10(3) ml g(-1) min(-1) (for striatum) and 2.2+/-0.4 x 10(3) ml g(-1) min(-1) (for cerebellum) in controls. Kin for AIB showed similar values. At 5 and 10 min RC periods, Kin for both substances increased in a non-specific manner in all brain regions studied. In the cortex, Kin for SPMD at 5 and 10 min RC periods were 3.2+/-0.4 x 10(3) and 2.9+/-0.3 x 10(3) ml g(-1) min(-1), respectively, and found to be maximum with respect to other brain regions studied. 30 and 60 min RC groups showed specific transport for SPMD, whilst there were no changes for Kin for AIB, in all brain regions studied. Hippocampus showed the maximum increase in Kin SPMD at 60 min RC (2.7+/-0.3 x 10(3) ml g(-1) min(-1)), corresponding to a percentage rise of 121%. Intraischemic mild brain hyperthermia (39 degrees C) gave rise to a striking increase in Kin at 60 min postischemia for both substances. These results suggest that there is a specific transport of SPMD into brain at 30 and 60 min RC periods following 20 min of forebrain ischemia. Moreover, dual functions of the BBB were perturbed with intracerebral mild hyperthermia during ischemia.
Assuntos
Barreira Hematoencefálica , Isquemia Encefálica/metabolismo , Hipotermia Induzida , Traumatismo por Reperfusão/metabolismo , Espermidina/farmacocinética , Ácidos Aminoisobutíricos/farmacocinética , Animais , Encéfalo/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
The Km and Vmax of [14C]-radiolabeled polyamines were determined for PC-3 and AT3B-1 cell lines. With PC-3 Km values are in the following order: ornithine> spermidine> spermine> putrescine, while with AT3B-1 it was spermidine> ornithine> spermine> putrescine. To determine which of these polyamines exhibit higher accumulation, the relative uptake of all the four amines was studied with prostate (PC-3, AT3B-1, LNCaP) and non-prostate (MCF-7, KLN-205, OVCAR) cell lines at 10 and 20 microM after 1 hour. Spermine and spermidine accumulated at higher levels in prostate (AT3B-1 and LNCaP) over non-prostate cell lines (p < 0.01). Putrescine accumulated more in PC-3 and LNCaP than the non-prostate cancer cells.
Assuntos
Radioisótopos de Carbono/farmacocinética , Poliaminas/farmacocinética , Neoplasias da Próstata/metabolismo , Animais , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/metabolismo , Humanos , Masculino , Ornitina/farmacocinética , Neoplasias da Próstata/diagnóstico por imagem , Putrescina/farmacocinética , Cintilografia , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espermidina/farmacocinética , Espermina/farmacocinética , Células Tumorais Cultivadas/diagnóstico por imagem , Células Tumorais Cultivadas/metabolismo , Neoplasias Urogenitais/diagnóstico por imagem , Neoplasias Urogenitais/metabolismoRESUMO
Smad7, a protein induced by transforming growth factor-beta1 (TGF-beta1) in many target cells, inhibits TGF-beta1 signal transduction and is thought to mediate an intracellular negative feedback response that limits TGF-beta1 effects. It is possible that overexpression of Smad7 could block specified effects of TGF-beta1 on mesangial cells, a TGF-beta target in glomerular disease. Smad7 mRNA was induced by TGF-beta1 within 1 h in a concentration-dependent manner in a transformed mouse mesangial cell (MMC) line. Uptake of (14)C-spermidine from the medium by MMC and the transcriptional activity of a segment of the human collagen pro-alpha2 type 1 chain (COL1A2) promoter fused to a luciferase reporter gene were used as indices of TGF-beta1. Treatment with TGF-beta1 increased (14)C-spermidine uptake rate in a time-, concentration-, and temperature-dependent manner. For example, exposure to 1 ng/ml TGF-beta1 for 15 h increased uptake approximately twofold, a response that was attenuated by cycloheximide. Transfection of Smad7 expression vector into MMC abrogated both TGF-beta1-dependent stimulation of spermidine uptake and COL1A2 promoter activity. It is concluded that: (1) TGF-beta1 induces Smad7 in MMC; (2) (14)C-spermidine uptake is a convenient quantitative index of TGF-beta1 effect in these cells; and (3) overexpression of Smad7 is a highly effective method of blocking at least some mesangial cell effects of TGF-beta1 that may warrant evaluation in vivo in experimental glomerular disease.
Assuntos
Proteínas de Ligação a DNA/farmacologia , Mesângio Glomerular/efeitos dos fármacos , Transativadores/farmacologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/farmacologia , Animais , Células Cultivadas , Colágeno/genética , Colágeno Tipo I , Cicloeximida/farmacologia , Proteínas de Ligação a DNA/genética , Mesângio Glomerular/citologia , Mesângio Glomerular/metabolismo , Humanos , Camundongos , Camundongos Transgênicos/genética , Regiões Promotoras Genéticas/fisiologia , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Proteína Smad7 , Espermidina/antagonistas & inibidores , Espermidina/farmacocinética , Transativadores/genética , Fator de Crescimento Transformador beta1RESUMO
In rat lung and cultured lung vascular cells, hypoxia decreases ornithine decarboxylase (ODC) activity and increases polyamine import. In this study, we used rat cultured pulmonary artery endothelial cells to explore the mechanism of hypoxia-induced reduction in ODC activity and determined whether this event was functionally related to the increase in polyamine import. Two strategies known to suppress proteasome-mediated ODC degradation, lactacystin treatment and use of cells expressing a truncated ODC incapable of interacting with the proteasome, prevented the hypoxia-induced decrease in ODC activity. Interestingly, though, cellular abundance of the 24-kDa antizyme, a known physiological accelerator of ODC degradation, was not increased by hypoxia. These observations suggest that an antizyme-independent ODC degradation pathway contributes to hypoxia-induced reductions of ODC activity. When reductions in ODC activity in hypoxia were prevented by the proteasome inhibitor strategies, hypoxia failed to increase polyamine transport. The induction of polyamine transport in hypoxic pulmonary artery endothelial cells thus seems to require decreased ODC activity as an initiating event.
Assuntos
Endotélio Vascular/enzimologia , Hipóxia/metabolismo , Ornitina Descarboxilase/metabolismo , Poliaminas/farmacocinética , Artéria Pulmonar/enzimologia , Animais , Western Blotting , Radioisótopos de Carbono , Bovinos , Células Cultivadas , Endotélio Vascular/citologia , Ativação Enzimática/fisiologia , Regulação Enzimológica da Expressão Gênica , Hipertensão Pulmonar/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/enzimologia , Ornitina Descarboxilase/genética , Proteínas/análise , Proteínas/metabolismo , Artéria Pulmonar/citologia , Putrescina/farmacocinética , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Espermidina/farmacocinética , Espermina/farmacocinéticaRESUMO
A series of novel spermine dimer analogues was synthesized and assessed for their ability to inhibit spermidine transport into MDA-MB-231 breast carcinoma cells. Two spermine molecules were tethered via their N(1) primary amines with naphthalenedisulfonic acid, adamantanedicarboxylic acid and a series of aliphatic dicarboxylic acids. The linked spermine analogues were potent polyamine transport inhibitors and inhibited cell growth cytostatically in combination with a polyamine synthesis inhibitor. Variation in the linker length did not alter polyamine transport inhibition. The amount of charge on the molecule may influence the molecular interaction with the transporter since the most potent spermidine transport inhibitors contained 5-6 positive charges.
Assuntos
Poliaminas/antagonistas & inibidores , Espermina/análogos & derivados , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas/química , Dimerização , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Poliaminas/farmacocinética , Espermidina/antagonistas & inibidores , Espermidina/farmacocinética , Espermina/química , Espermina/farmacologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Dimethylsilane tetramines are structural analogues of spermine with a (CH3)2 Si-group incorporated into the central carbon chain. They have potential as anticancer drugs. Their cytotoxic effect was considered to rely mainly on their polyamine antagonist property. In order to obtain new ideas about cellular mechanisms, which are potential targets of the dimethylsilane polyamines, the effects of these compounds on some basic cell functions, such as protein and DNA synthesis, and calmodulin antagonism were studied. In addition, their mode of accumulation in cells was investigated. It became evident that the intracellular accumulation of dimethylsilane polyamines is almost exclusively achieved via the polyamine transport system. However, the exchange of a part of the intracellular natural polyamines against dimethylsilane polyamines has only a small effect on polyamine uptake. Binding to the endoplasmic reticulum and inhibition of protein synthesis are presumably important for the cytotoxic action of bis(11-amino-4,8-diazaundecyl)dimethylsilane, a hexamine, but seem of no importance for the tetramines. Calmodulin antagonism, however, is likely to contribute to their cytotoxic effect.
Assuntos
Antineoplásicos/farmacologia , Antineoplásicos/farmacocinética , Calmodulina/análogos & derivados , Poliaminas/farmacologia , Poliaminas/farmacocinética , Silanos/farmacologia , Silanos/farmacocinética , Animais , Células CHO/efeitos dos fármacos , Células CHO/metabolismo , Calmodulina/metabolismo , Agregação Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Cricetinae , DNA de Neoplasias/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Leucina/antagonistas & inibidores , Leucina/metabolismo , Leucemia L1210/tratamento farmacológico , Leucemia L1210/metabolismo , Metenamina/farmacocinética , Metenamina/farmacologia , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Ratos , Espermidina/antagonistas & inibidores , Espermidina/farmacocinética , Timidina/antagonistas & inibidores , Timidina/metabolismo , Poliamina OxidaseRESUMO
1. The gamma subunit is a specific component of the plasmalemmal Na(+),K(+)-ATPase. Like structurally related single-spanning membrane proteins such as cardiac phospholemman, Mat-8 and renal CHIF, large ion conductances are activated when gamma subunits are expressed in Xenopus oocytes. 2. Here we report critical properties of the gamma-activated conductance. The gamma-activated conductance showed non-selective cationic and anionic permeation, and extremely slow kinetics, with an activation time constant > 1 s following steps to -100 mV. 3. The gamma-activated conductance was inhibited by extracellular divalent ions including Ba(2+) (K(i) = 0.7 mM) and Ca(2+) (K(i) = 0.4 mM). 4. 2-Deoxyglucose (MW approximately 180), inulin (MW approximately 5000) and spermidine (MW approximately 148) efflux could occur through the gamma-activated conductance pathway, indicating a large pore diameter. In contrast, dextran-70 (MW approximately 70 000) did not pass through the gamma-activated channel, indicating an upper limit to the pore size of approximately 50 A (5 nm). 5. Similar conductances that are permeable to large molecules were activated by extreme hyperpolarization (> -150 mV) of uninjected oocytes. 6. We conclude that the Na(+),K(+)-ATPase gamma subunits activate Ca(2+)- and voltage-gated, non-selective, large diameter pores that are intrinsically present within the oocyte membrane.