RESUMO
trans-Urocanic acid (UCA) acts as a chromophore for UV radiation in the epidermis and isomerizes to cis-UCA which then initiates some of the changes leading to UV-induced immunosuppression. The mechanism of the immunomodulation by cis-UCA is unknown at present, but one possibility is that the interaction between cis-UCA and keratinocytes causes the release of immunosuppressive cytokines locally. To test this hypothesis, PAM-212 cells, a murine keratinocyte cell line, were incubated with 0.10-100 micrograms/mL trans- and cis-UCA for 6 or 24 h, respectively. The expression of interleukin (IL)-10, transforming growth factor (TGF)-beta and tumor necrosis factor (TNF)-alpha messenger RNA (mRNA) was then measured by reverse transcription-polymerase chain reaction in comparison with the mRNA for the house-keeping gene, beta-actin. No change or significant induction of any of the cytokine messages occurred. However, the expression of IL-10 messenger RNA (mRNA) was induced 24 h after UVB irradiation (300 J/m2) and that of TNF-alpha mRNA occurred 6 h after treatment with phorbol myristate acetate. The expression of IL-10 protein was also examined by immunostaining in both PAM-212 cells and B16-F10 murine melanoma cells between 3 and 48 h after incubation with 10 and 100 micrograms/mL cis- and trans-UCA. No alteration was seen with either isomer at either concentration. In contrast, UVB irradiation of both cell lines resulted in a marked increase in intracellular IL-10 protein at 24 and 48 h. Therefore the upregulation of the immunosuppressive cytokines, IL-10, TNF-alpha and TGF-beta, in keratinocytes is unlikely to be the mechanism by which cis-UCA induces immunosuppression in mice.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-10/genética , Queratinócitos/efeitos dos fármacos , Fator de Crescimento Transformador beta/genética , Fator de Necrose Tumoral alfa/genética , Ácido Urocânico/farmacologia , Animais , Sequência de Bases , Primers do DNA , Queratinócitos/citologia , Camundongos , RNA Mensageiro , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
The binding of unfractionated heparin to endothelium is thought to be responsible for the rapid and saturable phase of unfractionated heparin clearance. Thrombin can induce endothelial cells to express and/or secrete a number of heparin binding proteins that have the potential to increase the binding of unfractionated heparin and to a lesser extent the binding of low molecular weight heparin. To explore this possibility, we examined the binding of unfractionated heparin and low molecular weight heparin to thrombin-activated endothelial cells. Cultured human umbilical vein endothelial cells were used to determine the binding of 125I-labeled unfractionated heparin and low molecular weight heparin to untreated and to thrombin-activated cells. After thrombin treatment, we obtained a time-dependent increase in the binding of radio-labeled unfractionated heparin. In contrast, there was much less binding of low molecular weight heparin, and a time-dependent increase was not apparent. After 30, 45, and 60 minutes of thrombin treatment, the binding of unfractionated heparin was significantly higher than that of low molecular weight heparin. The increase in binding of unfractionated heparin to thrombin-activated cells also was demonstrated using fluorescently labeled unfractionated heparin followed by fluorescence microscopy. The average fluorescence intensity of thrombin-treated cells increased by 44% when compared with resting cells. The present results indicate that thrombin can increase the binding of unfractionated heparin to human umbilical vein endothelial cells. Thus, an activated endothelium may contribute to the variability of the anticoagulant response to unfractionated heparin. In contrast, the binding of low molecular weight heparin is much less affected, which may account for its better bioavailability and longer half-life.
Assuntos
Endotélio Vascular/citologia , Heparina de Baixo Peso Molecular/metabolismo , Heparina/metabolismo , Trombina/metabolismo , Sítios de Ligação , Endotélio Vascular/ultraestrutura , Humanos , Radioisótopos do Iodo , Microscopia de Fluorescência , Trombina/farmacologia , Cordão Umbilical/citologiaRESUMO
Previously, we demonstrated in a rat model of heparin-induced osteoporosis that low molecular weight heparin (LMWH) produces less bone loss than unfractionated heparin, and that only heparin increases osteoclast number and activity. In contrast, both heparin and LMWH were found to decrease osteoblast function to a similar extent, possibly because at the doses tested both agents produced maximal inhibition. To examine the relative effects of heparin and LMWH on osteoblast function more closely we used an in vitro bone nodule assay, together with measurements of alkaline phosphatase (ALP) activity. Both agents inhibited bone nodule formation and ALP activity in a concentration-dependent manner, but 6 to 8-fold higher concentrations of LMWH were required to achieve equivalent effects. The effect of heparin on osteoblast function was both chain-length and negative charge-dependent because the ability of defined heparin fragments to inhibit nodule formation correlated with their molecular weight (r = 0.98), and N-desulfated heparin was less inhibitory than heparin. In contrast, the effect of heparin on osteoblast function was pentasaccharide-independent because heparin with low affinity for antithrombin had similar activity to heparin with high antithrombin activity. These findings help to explain mounting clinical evidence that the risk of osteoporosis is lower with LMWH than with heparin.
Assuntos
Reabsorção Óssea/induzido quimicamente , Fibrinolíticos/farmacologia , Heparina de Baixo Peso Molecular/farmacologia , Heparina/farmacologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Fibrinolíticos/efeitos adversos , Heparina/efeitos adversos , Heparina de Baixo Peso Molecular/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , RatosRESUMO
We have previously demonstrated that the nonspecific binding of unfractionated heparin (UFH) to plasma proteins has a marked modulating effect on its anticoagulant activity. Since some heparin-binding proteins are also acute-phase-reactant proteins, we explored the possibility that the induction of the acute-phase response can increase the plasma concentrations of heparin-binding proteins. The recovery of a fixed amount of UFH or low-molecular-weight heparin (LMWH) added in vitro to rat plasma samples obtained at various time intervals after the administration of intravenous endotoxin or subcutaneous turpentine was compared with that of saline-treated control animals. The anti-factor Xa activity was measured in the plasma samples before and after the addition of a chemically modified low-affinity heparin (LAH) to displace the proportion of the added heparin that is reversibly bound to plasma proteins. Our results show that at 6 hours post-endotoxin and at 24 hours post-turpentine treatment, virtually no anti-factor Xa activity could be measured in the plasma samples, while the expected levels were obtained for control plasma. After the addition of LAH to displace protein-bound UFH, essentially the same anti-factor Xa levels were measured in the plasma from all three treatment groups. These results indicate that induction of the acute-phase reaction can dramatically increase the levels of heparin-binding proteins in rat plasma. In addition, we compared the anti-factor Xa recovery of UFH with that of an LMWH from the plasma of endotoxin- and saline-treated rats and demonstrated that LMWH binds less to plasma proteins than UFH, even in plasma in which the levels of heparin-binding proteins are markedly elevated. The recovery of a fixed amount of UFH added in vitro to human plasma from septic patients was also reduced, but not to the same extent as seen in rat plasma. Removal of candidate heparin-binding and acute-phase proteins by immunodepletion indicated that vitronectin plays an important role in the nonspecific binding of UFH in patient plasma.
Assuntos
Reação de Fase Aguda/sangue , Reação de Fase Aguda/etiologia , Proteínas de Transporte/sangue , Heparina/sangue , Animais , Western Blotting , Endotoxinas/farmacologia , Heparina de Baixo Peso Molecular/sangue , Humanos , Depleção Linfocítica , Masculino , Ratos , Ratos Sprague-Dawley , Sepse/sangue , Terebintina/farmacologia , Vitronectina/sangueRESUMO
The serum selenium (Se) concentrations of apparently healthy 20-40 year old blood donors from different parts of Switzerland were determined by electrothermal atomic absorption spectrometry (GFAAS). Application of a rhodium/magnesium matrix-modifier resulted in improved performance in comparison with a palladium modifier. The method was validated by hydride ICP-MS and quality-controlled by independent analysis using GFAAS with palladium as matrix-modifier; no bias was detected. The serum Se concentrations for male (n = 387) and female (n = 243) subjects fell into a normal distribution with mean values and standard deviations of 96.0 +/- 13.3 micrograms/L (1.22 +/- 0.17 mumol/L) and 87.9 +/- 14.4 micrograms/L (1.11 +/- 0.18 mumol/L), respectively. These values corresponded well to the formerly estimated mean daily intakes. Small but significant differences in mean serum Se concentrations were found between genders, ethnic groups as well as geographic regions, whereas age had no influence. The overall Se status of the Swiss population is assessed as adequate, somewhat higher than in the countries adjacent to Switzerland, but lower than in the U.S.A. or Finland. There is no evidence that the Se status of the population has changed over the past 10 years. However, it appears that some Swiss population groups may have a borderline Se status.
Assuntos
Dieta , Selênio/sangue , Espectrofotometria Atômica/métodos , Adulto , Feminino , Humanos , Modelos Lineares , Masculino , Controle de Qualidade , Valores de Referência , Reprodutibilidade dos Testes , Distribuição por Sexo , SuíçaRESUMO
Ultraviolet radiation can induce the transcription and release of cytokines from keratinocytes (KC's). These cytokines have the potential to modulate local and systemic immunologic responses. In this paper we report that northern blotting showed that human KC and KC lines expressed a 1.2-1.4 kb transcript for the chemokine and melanoma growth-stimulatory protein, GRO-alpha and that ultraviolet B radiation (UVB) could upregulate the expression of GRO-alpha mRNA and protein in the KC line A431. The GRO-alpha gene response to UVB was maximal at 48h post-irradiation with 70 J/m2. Reverse transcription-polymerase chain reaction (RT-PCR) revealed a 4.5-fold increase in GRO-alpha mRNA over basal levels (p < 0.001). GRO-alpha protein was measured in the culture media by enzyme-linked immunosorbent assay (ELISA). Media from unirradiated cultures contained 1166 +/- 83 pg/ml GRO-alpha protein. After UVB, a time-dependent increase in GRO-alpha protein was seen in the culture media from 6-48h. At 48h post-irradiation the GRO-alpha protein content was 27583 +/- 678 pg/ml, or 23 times the basal level. This protein release could be inhibited by 70% when the cells were pre-incubated with 10 micrograms/ml interleukin-1 receptor antagonist (IL-1RA). We also show that another potent leukocyte chemoattractant, Interleukin-8 (IL-8), was induced in A431 cells by UVB. This induction of IL-8 mRNA began as early as 3h post-irradiation, when it reached twice basal levels (p < 0.05) and reached 4.5-fold basal levels at 48h post-irradiation (p < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Quimiocinas CXC , Fatores Quimiotáticos/biossíntese , Regulação da Expressão Gênica , Substâncias de Crescimento/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular , Interleucina-8/biossíntese , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Sequência de Bases , Células Cultivadas , Quimiocina CXCL1 , Fatores Quimiotáticos/genética , Fatores Quimiotáticos/imunologia , Ensaio de Imunoadsorção Enzimática , Substâncias de Crescimento/genética , Substâncias de Crescimento/imunologia , Humanos , Interleucina-1/farmacologia , Interleucina-8/genética , Interleucina-8/imunologia , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Fatores de Tempo , Células Tumorais Cultivadas , Raios UltravioletaRESUMO
Cyclosporine A (CsA) has been shown to be an effective therapeutic agent for a wide variety of cutaneous diseases yet its exact mechanism of action is still unclear, although one well-defined effect of CsA is the inhibition of T-cell-derived cytokine expression. We recently demonstrated in vitro that CsA inhibits cell proliferation and suppresses cytokine gene expression in keratinocytes. In this study, we report the in vivo effects of CsA on skin cytokine gene expression as determined by reverse-transcriptase polymerase chain reaction. C57BL6 mice (female, 8-10 weeks old) were subcutaneously injected with CsA in olive oil (0, 5 and 10 mg/kg) every other day for 3 weeks. Treatment with 5 mg/kg CsA inhibited both interleukin (IL)-1 alpha and tumor necrosis factor alpha gene expression by about 70 and 90%, respectively, relative to vehicle control levels. However, IL-6 gene expression did not significantly change. Injection of 10 mg/kg CsA inhibited expression of all three genes by 80-90% relative to control levels. These data show that CsA can inhibit constitutive cytokine gene expression in mouse skin.
Assuntos
Ciclosporina/farmacologia , Citocinas/biossíntese , Citocinas/genética , Expressão Gênica/efeitos dos fármacos , Pele/metabolismo , Animais , Sequência de Bases , Ciclosporina/administração & dosagem , Depressão Química , Feminino , Injeções Subcutâneas , Interleucina-1/biossíntese , Interleucina-1/genética , Interleucina-6/biossíntese , Interleucina-6/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Pele/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
A protein purified from Eschericheri coli has previously been shown to have cytotoxic effects on neoplastic cells of several lineages both in vitro and in vivo. Accordingly, this protein has been named anti-neoplastic protein (ACP). Although ACP kills neoplastic cells by inducing apoptosis, it has negligible effects on various normal cells. In addition to the direct cytotoxic effects of ACP on tumour cells, previous studies have shown that in vivo ACP increases tumouricidal activity of cytotoxic lymphocytes. We investigated whether cytokines from host or tumour cells play a part in the enhanced cellular immunity seen in ACP-treated tumour-bearing mice. Growth of normal human keratinocytres (KC) was not significantly affected by subnanogram amounts of ACP, however ACP dose-dependently killed KHT cells, a murine fibrosarcoma cell line (LD50 = 8 x 10(4) ng/cell). as well as the human squamous carcinoma cell line COLO-16 (LD50 = 2.5 x 10(-4) ng/cell). Testing purified ACP on cultures of normal keratinocytes and squamous carcinoma cell lines revealed that ACP could induce both mRNA and protein for interleukin-6 (IL-6). Messenger RNA for IL-6 increased dose-dependently 4h after treatment of COLO-16 squamous carcinoma cells with 10(-4) to 10(-2) ng/cell ACP. Maximal increment was 50-fold. Interleukin-6 message remained elevated up to 24h later in both normal keratinocytes and squamous carcinoma cultures treated with ACP. Conditioned supernates from these cultures were analysed by ELISA and found to have 4-fold higher levels of IL-6 protein than untreated cells after 4h. After 24h, IL-6 did not increase above the 4h level. Boiling of the ACP preparation showed that the cytokine induction was not due to contaminating lipopolysaccharide. The cytoxic effect of ACP on tumour cells in vitro was not due to IL-6 protein induction since neither recombinant IL-6, nor the other proinflammatory cytokines, IL-1 alpha or Tumour necrosis factor-alpha (TNF-alpha) (0.1-10ng/ml) were able to kill malignant cells. We demonstrated IL-6 gene induction by ACP in the squamous carcinoma lines as well as in normal KC. This suggests that the in vivo effectiveness of ACP against tumours may be due to stimulatory effects of IL-6 on host immunity.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Bactérias/farmacologia , Carcinoma de Células Escamosas/metabolismo , Interleucina-6/biossíntese , Queratinócitos/efeitos dos fármacos , Humanos , Queratinócitos/metabolismo , Lipopolissacarídeos/farmacologia , Proteínas Recombinantes/farmacologia , Células Tumorais CultivadasRESUMO
The cDNA sequence of the 60 kDa heat-shock protein from mouse 3T3 cells has been determined. The deduced amino acid sequence of mouse hsp60 protein differs from the corresponding proteins from Chinese hamster and human cells in 7 and 13 residues, respectively, most of which are conservative replacements.
Assuntos
Proteínas de Choque Térmico/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Chaperonina 60 , Cricetinae , DNA/genética , Escherichia coli/genética , Humanos , Camundongos , Dados de Sequência Molecular , Mycobacterium leprae/genética , Saccharomyces cerevisiae/genéticaRESUMO
A number of clones that specifically hybridize to the human hsp60 cDNA (chaperonin protein; GroEL homolog) were isolated from human and Chinese hamster ovary cell genomic libraries. DNA sequence analysis shows that one of these clones, pGem-10, is completely homologous to the human hsp60 cDNA (in both coding and noncoding regions) with no intervening sequences. The other human clones analyzed were all nonfunctional pseudogenes containing numerous small additions, deletions, and base substitutions, but no introns. On the basis of sequence data, six different hsp60 pseudogenes were identified in human cells. In addition, we also cloned and completely sequenced a genomic clone from CHO cells. This clone, which was also a pseudogene, contained a small 87-nucleotide intron near the 3' end. Southern blot analysis of human, mouse, and Chinese hamster DNA, digested with unique restriction enzymes (no sites in cDNA), indicates the presence of about 8-12 genes for hsp60 in the vertebrate genomes. The sequence data, however, suggest that most of these genes, except one (per haploid genome), are likely to be nonfunctional pseudogenes.
Assuntos
Proteínas de Choque Térmico/genética , Animais , Sequência de Bases , Chaperonina 60 , Cricetinae , Cricetulus , Biblioteca Gênica , Biblioteca Genômica , Humanos , Dados de Sequência Molecular , Família Multigênica , PseudogenesRESUMO
A major cellular protein (P2; approximately 70 kilodaltons) which is altered in Chinese hamster ovary (CHO) cell mutants resistant to the microtubule inhibitors colchicine and podophyllotoxin has been shown to correspond to the constitutive form of the 70-kilodalton heat shock protein (hsc70). The inference that P2 and hsc70 are the same protein is based on the following observations: (i) migration of P2 in two-dimensional polyacrylamide gels in the same position as that reported for hsc70; (ii) cross-reactivity of a monoclonal antibody which reacts with both the constitutive and induced forms of hsp70 with the P2 spot from wild-type CHO cells and with both P2 and a mutant form of P2 in a CHO cell mutant; (iii) specific reactivity of a polyclonal antibody to P2 with both the constitutive and heat-induced forms of hsp70 in human cells; (iv) identical immunofluorescent staining of dot/patchlike structures with both P2 and hsp70 antibodies in human and CHO cells; and (v) a cDNA clone for hsc70 has been isolated and sequenced from wild-type CHO cells. The in vitro transcription and translation product of this cDNA has been shown to comigrate with the P2 protein spot in two-dimensional gels, indicating their identity. The fact that there is an alteration in hsc70 in mutants resistant to antimitotic drugs suggests a role for this protein in the in vivo assembly and function of microtubules.
Assuntos
Proteínas de Choque Térmico/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Sequência de Bases , Linhagem Celular , Chaperoninas , Clonagem Molecular , Cricetinae , Cricetulus , Resistência a Medicamentos , Eletroforese em Gel Bidimensional , Feminino , Imunofluorescência , Dados de Sequência Molecular , Podofilotoxina/farmacologia , Proteínas/fisiologiaRESUMO
Using antibodies to a mitochondrial molecular chaperone class of protein, which is specifically altered in mutants resistant to microtubule (MT) inhibitors, the effect of a number of MT and mitochondrial inhibitors on the cellular distribution of mitochondria and various cytoskeletal filaments was examined. Treatment of Chinese hamster ovary (CHO) or chicken embryo fibroblast (CEF) cells with the MT inhibitors podophyllotoxin, colchicine, nocodazole and vinblastine caused depolymerization of cellular MTs, but had no significant effect on the distribution patterns of mitochondria. This is attributed to the association of mitochondria with intermediate filaments (IFs) which are not destroyed under these conditions. In contrast to MT inhibitors, treatment of CEFs with the potassium ionophores nonactin and valinomycin caused aggregation of mitochondria towards the perinuclear region of the cells, without having any apparent effect on cellular MTs. This observation suggests that mitochondrial membrane potential, which is abolished by these drugs, play a role in the cellular distribution of mitochondria. In cells recovering from the effects of MT inhibitors, mitochondria have been found to surround the MT organizing complexes and upon complete recovery a realignment of MTs with mitochondria takes place. These observations suggest that MT growth in cells does not occur in a completely random manner but that mitochondria may play some role in their directional growth.
Assuntos
Antineoplásicos/farmacologia , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Animais , Linhagem Celular , Embrião de Galinha , Colchicina/farmacologia , Imunofluorescência , Cobaias , Microtúbulos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mutação , Nocodazol/farmacologia , Podofilotoxina/farmacologia , Vimblastina/farmacologiaRESUMO
In Chinese hamster ovary cells, stable mutants exhibiting specific resistance or collateral sensitivity towards the various microtubule inhibitors podophyllotoxin, colchicine, griseofulvin, taxol, nocodazole, vinblastine, and maytansine have been isolated. A number of independent mutants selected for resistance to podophyllotoxin and colchicine contain electrophoretically altered forms of two proteins, P1 and P2, of relative molecular masses of approximately 63 000 and 69 000, respectively. The proteins P1 and P2 have been shown to be microtubule related by a number of different genetic and biochemical criteria and are among the major proteins of Chinese hamster ovary cells, being present in approximately equimolar amounts with tubulin. In addition, a griseofulvin-resistant mutant contains a novel mutation (presumably nonsense) which reduces the relative molecular mass of a protein, P5 (relative mass congruent to 200 000), by about 20 000. Specific antibodies to protein P1 have been raised and cross-reactivity studies show that a similar protein is also present in cells from all vertebrate species examined, viz. man, monkey, mouse, Chinese hamster, Syrian hamster, and chicken. Immunofluorescence studies with anti-P1 and anti-tubulin antibodies show that, in interphase cells from various species, the P1 antibody reacts specifically with mitochondria whose overall cellular distribution is strikingly similar to the distribution of microtubules. The mitochondrial localization of the microtubule-related protein P1 provides strong suggestive evidence regarding the existence of a chemical and functional linkage between these two structures, with protein P1 playing an important role in this linkage. Some implication of these results are discussed and it is suggested that mitochondria play an important role in the dynamics of microtubules.