RESUMO
The structural and magnetic properties of a collection of nanoparticles coated by Poly(methyl methacrylate) through a wet chemical synthesis have been investigated. The particles display either an amorphous (M = Fe, Co) M-B arrangement or a mixed structure bcc-Fe and fcc-Co + amorphous M-B. Both show the presence of a metal oxi-hydroxide formed in aqueous reduction. The organic coating facilitates technological handling. The cost-effective synthesis involves a reduction in a Poly(methyl methacrylate) aqueous solution of iron(II) or cobalt(II) sulphates (< 0.5 M) by sodium borohydride (< 0.5 M). The particles present an oxidized component, as deduced from X-ray diffraction, Mössbauer and Fe- and Co K-edge X-ray absorption spectroscopy and electron microscopy. For the ferrous alloys, this Fe-oxide is alpha-goethite, favoured by the aqueous solution. The Poly(methyl methacrylate) coating is confirmed by Fourier transform infrared spectroscopy. In pure amorphous core alloys there is a drastic change of the coercivity from bulk to around 30 Oe in the nanoparticles. The mixed structured alloys also lie in the soft magnetic regime. Magnetisation values at room temperature range around 100 emu/g. The coercivity stems from multidomain particles and their agglomeration, triggering the dipolar interactions.
RESUMO
To determine the biochemical basis for reduced PGE2 expression by cord blood monocytes relative to adult peripheral blood monocytes, fatty acid composition of cell membranes together with the amount and activity of enzymes involved in the cyclooxygenase pathway were investigated. Gas chromatographic analysis of mononuclear cell fatty acids showed a significant reduction in long chain fatty acids, including arachidonic acid. Acylase and phospholipase A2 activity, together with lipocortin 1 levels, were similar in cord and adult monocytes. No difference in amounts of the 70-kDa cyclooxygenase type 1 enzyme was observed between the monocyte sources. The 74k-Da cyclooxygenase type 2 enzyme was synthesized de novo upon LPS stimulation in comparable amounts in both cord and adult monocytes, with both monocyte types responding with equal sensitivity to LPS. The ability of microsomes, prepared from cord monocytes, to convert arachidonic acid into eicosanoids of the cyclooxygenase pathway was reduced. In contrast, the ability of acetylated microsomes to convert PGG2 to PGE2 was comparable, suggesting that the deficiency in PGE2 production by cord monocytes is due to reduced activity, specifically at the cyclooxygenase-active site rather than the peroxidase active site. In conclusion, it appears that both reduced fatty acid content and diminished cyclooxygenase activity are responsible for diminished eicosanoid production by cord monocytes.
Assuntos
Dinoprostona/biossíntese , Ácidos Graxos/metabolismo , Recém-Nascido/metabolismo , Isoenzimas/metabolismo , Lipídeos de Membrana/metabolismo , Monócitos/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Acetilação , Adulto , Amidoidrolases/metabolismo , Anexina A1/metabolismo , Ácidos Araquidônicos/metabolismo , Dinoprostona/genética , Sangue Fetal/citologia , Humanos , Microssomos/enzimologia , Fosfolipases A/metabolismo , Fosfolipases A2RESUMO
Umbilical cord blood and adult peripheral blood monocytes were separated into two subpopulations, based on the intensity of CD14 expression, and the coexpression of various antigens associated with monocyte function was examined. The majority of cord and adult monocytes expressed CD14 at a high density (CD14bright) while approximately 15% of monocytes expressed this antigen at a lower level (CD14dim). Three times as many CD14dim monocytes expressed CD16 (Fc gamma RIII) as did CD14bright monocytes in both cord and adult preparations, while its level of expression (mean fluorescence intensity) was significantly reduced on cord CD14dim monocytes relative to adult CD14dim monocytes. The percentage of cord blood CD14dim monocytes expressing human leucocyte antigen-DR (major histocompatibility complex class II antigen) was significantly reduced relative to adult CD14dim cells; however, the level of expression of this antigen was greater on both cord and adult CD14dim monocytes compared with CD14bright cells. Cord CD14bright cells expressed CD36 (OKM5) to a greater extent than did their adult counterparts. The level of expression of CD62L was reduced on cord CD14dim cells compared with adult CD14dim cells. CD11b (CR3) was expressed both on a higher percentage of cells and also with a greater intensity on both cord and adult CD14bright monocytes compared with their CD14dim counterpart. These results show that significant differences exist between cord and adult monocyte subpopulations with regard to expression of various antigens associated with specific effector function.
Assuntos
Sangue Fetal/imunologia , Receptores de Lipopolissacarídeos/imunologia , Monócitos/imunologia , Adulto , Antígenos CD36/análise , Antígenos CD36/imunologia , Citometria de Fluxo , Imunofluorescência , Antígenos HLA-DR/análise , Antígenos HLA-DR/imunologia , Humanos , Recém-Nascido , Selectina L/análise , Selectina L/imunologia , Receptores de Lipopolissacarídeos/análise , Antígeno de Macrófago 1/análise , Antígeno de Macrófago 1/imunologiaRESUMO
PGE2, which is produced in large amounts by monocytes, is considered to be an important autocrine factor in the regulation of the immune response. Altered sensitivity to PGE2 is known to reflect the differentiation status of monocytes/macrophages. Since newborn monocytes are thought to be less differentiated than adult peripheral blood monocytes, this study was designed to determine whether the diminished newborn monocyte function, relative to adult peripheral blood monocytes, might be the result of an altered autocrine responsiveness to PGE2. Phosphodiesterase activity as well as cAMP and TNF alpha levels in response to PGE2 were measured in newborn and adult monocytes, as an index of sensitivity to PGE2. Basal intracellular cAMP levels were reduced in cord monocytes compared with adult monocytes. Addition of increasing concentrations of PGE2 (10M(-10)-10M-6) caused similar fold increases in intracellular levels of cAMP for both populations. Lipopolysaccharide-induced TNF alpha production was significantly reduced in a similar fashion for both cord and adult monocytes at high (> or = 10(-7)M) PGE2 concentration. Phosphodiesterase activity was comparable in lysates from both populations. This study shows that PGE2 does not differentially influence newborn and adult monocyte responses and therefore that the diminished functional abilities of newborn monocytes is not due to altered autocrine responsiveness.
Assuntos
Envelhecimento/imunologia , Dinoprostona/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Adulto , Envelhecimento/sangue , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , AMP Cíclico/biossíntese , AMP Cíclico/sangue , Dinoprostona/fisiologia , Humanos , Recém-Nascido , Leucócitos Mononucleares/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Interleukin-12 (IL-12) production by human monocytes is stringently regulated through the inducibility of both subunits, p35 and p40, and expression of p35 mRNA is the limiting factor for the secretion of the bioactive IL-12 p70 heterodimer. Optimal induction of p35 mRNA requires priming of the monocytes by interferon-gamma (IFN-gamma), followed by brief exposure to lipopolysaccharide or other bacterial products. To investigate control of p35 gene expression, we isolated genomic clones containing the human p35 gene and determined the 5' end of the mRNA expressed in monocytes. We discovered that a unique p35 transcript is induced in monocytes that begins downstream of a consensus TATA box that lies within the 5' end of the cDNA originally cloned from Epstein-Barr virus (EBV)-transformed B cells. Analysis of p35 mRNA by Northern blotting showed that the message from monocytes is approximately 200 bases shorter than message derived from the EBV-transformed B-cell line VDS. The initiation sites downstream from the TATA box were confirmed by RNase protection and 5' RACE. The data indicate that p35 transcription can initiate from different sites depending on the cell type and that the shorter inducible transcript in monocytes is the one that accumulates after stimulation. Protein translation of these two forms may result in proteins of different sizes with potential implications for the regulation of IL-12 secretion and function.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Herpesvirus Humano 4 , Interferon gama/farmacologia , Interleucina-12/biossíntese , Interleucina-12/genética , Linfócitos/patologia , Linfócitos/fisiologia , Monócitos/fisiologia , Regiões Promotoras Genéticas/genética , Sequência de Bases , Linhagem Celular Transformada , Transformação Celular Viral , Humanos , Linfócitos/virologia , Dados de Sequência Molecular , Monócitos/patologia , TATA BoxRESUMO
IL-12 is a heterodimeric cytokine produced by APC that critically regulates cell-mediated immunity. Because of its crucial function during immune responses, IL-12 production is stringently regulated, in part through transcriptional control of its p35 subunit, which requires the differentiative effects of IFN-gamma for expression. To determine whether post-transcriptional aspects of IL-12 production might be regulated, we examined intracellular protein processing of each subunit. We report here that p40 and p35 subunits are processed by disparate pathways. Whereas processing of p40 conforms to the cotranslational model of signal peptide removal concomitant with translocation into the endoplasmic reticulum (ER), processing of p35 does not. Translocation of the p35 preprotein into the ER was not accompanied by cleavage of the signal peptide; rather, removal of the p35 signal peptide occurred via two sequential cleavages. The first cleavage took place within the ER, and the cleavage site localized to the middle of the hydrophobic region of the signal peptide. Although the preprotein was glycosylated upon entry into the ER, its glycosylation status did not affect primary cleavage. Subsequently, the remaining portion of the p35 signal peptide was removed by a second cleavage, possibly involving a metalloprotease, concomitant with additional glycosylation and secretion. Secretion could be inhibited by mutation of the second cleavage site or by inhibition of glycosylation with tunicamycin. In contrast, p40 secretion was not affected by inhibition of glycosylation. Our findings demonstrate that IL-12 subunits are processed by disparate pathways and suggest new modalities for regulation of IL-12 production.
Assuntos
Interleucina-12/metabolismo , Líquido Intracelular/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/imunologia , Sinais Direcionadores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Glicosilação , Humanos , Hidrólise , Interleucina-12/química , Substâncias Macromoleculares , Modelos Biológicos , Modelos Químicos , Dados de Sequência Molecular , Peso Molecular , Biossíntese de Proteínas/imunologia , Precursores de Proteínas/química , Sinais Direcionadores de Proteínas/químicaRESUMO
In this report we show that mithramycin considerably increases the direct cytotoxic effect of tumour necrosis factor (TNF) on tumour cells in vitro. Sensitisation to TNF-induced apoptosis was prevented by the broad caspase inhibitor zVAD-fmk, whereas overexpression of Bcl-2 had no effect. Mithramycin also potentiated cell death induced by Fas agonistic antibodies. In contrast, mithramycin reduced the percentage of cells undergoing apoptosis due to factor withdrawal. TNF-induced activation of NF-kappaB (NF-kappaB)-dependent gene expression was not modulated by mithramycin treatment. Concomitantly with the increased sensitivity, the protein level of the short-spliced cFLIP variant was downregulated. These results indicate that mithramycin enhances TNF-induced cell death in an NF-kappaB-independent manner, and suggest that the Fas-associated death domain protein plays a crucial role in the TNF-sensitising effect of mithramycin.