RESUMEN
The steady state and time-resolved fluorescence and infrared (IR) properties of 4- and 5-cyanotryptophan (CNTrp) are investigated and compared, and the tryptophan (Trp) analogs are found to be very attractive to study structural and dynamic properties of proteins. The position of the nitrile substitution as well as the solvent environment influences the spectroscopic properties (solvatochromism). Similar to native Trp, electronic (nanosecond) lifetime and emission spectra are modulated by the environment, making CNTrps attractive fluorescent probes to study the structural dynamics of proteins in complex media. The nitrile absorption in the IR region can provide local structural information as it responds sensitively to changes in electrostatics and hydrogen bond (HB) interactions. Importantly, we find that 4CNTrp exhibits a single absorption in the nitrile stretch region, while the model compound 4CN-indole (4CNI) shows two. Even though the spectrum of the model compound is perturbed by a Fermi resonance, we find that 4CNTrp itself is a useful IR label. Moreover, if the nitrile group is substituted at the 5 position, the Trp analog predominantly reports on its HB status. Because the current literature on similar compounds is too limited for a detailed solvatochromic analysis, we extend the available data significantly. Only now are microscopic details such as the mentioned sensitivity to electrostatics coming to light. The vibrational lifetime of the CN moiety (acting on a picosecond time scale in contrast to the nanosecond time scale for fluorescent emission) allows for its application in 2D-IR spectroscopy in the low picosecond range. Taken together, the benefits of CNTrps are that they absorb and emit separately from the naturally occurring Trp and that in these dual fluorescence/vibrational labels, observables of IR and fluorescence spectroscopy are modulated differently by their surroundings. Because IR absorption and fluorescence operate on different time and length scales, they thus provide complementary structural information.
Asunto(s)
Colorantes Fluorescentes/química , Sondas Moleculares/química , Nitrilos/química , Triptófano/análogos & derivados , Triptófano/química , Fluorescencia , Indoles/química , Estructura Molecular , Espectrometría de Fluorescencia , Espectrofotometría InfrarrojaRESUMEN
The apoflavodoxin protein from Azotobacter vinelandii harboring three tryptophan (Trp) residues, was biosynthetically labeled with 5-fluorotryptophan (5-FTrp). 5-FTrp has the advantage that chemical differences in its microenvironment can be sensitively visualized via (19)F NMR. Moreover, it shows simpler fluorescence decay kinetics. The occurrence of FRET was earlier observed via the fluorescence anisotropy decay of WT apoflavodoxin and the anisotropy decay parameters are in excellent agreement with distances between and relative orientations of all Trp residues. The anisotropy decay in 5-FTrp apoflavodoxin demonstrates that the distances and orientations are identical for this protein. This work demonstrates the added value of replacing Trp by 5-FTrp to study structural features of proteins via (19)F NMR and fluorescence spectroscopy.
Asunto(s)
Apoproteínas/química , Flavodoxina/química , Triptófano/análogos & derivados , Apoproteínas/genética , Azotobacter vinelandii/química , Flavodoxina/genética , Polarización de Fluorescencia/métodos , Flúor/química , Espectroscopía de Resonancia Magnética/métodos , Resonancia Magnética Nuclear Biomolecular , Triptófano/química , Triptófano/genéticaRESUMEN
Two types of defect between femoral hip implants and cement have been identified. Interfacial porosity arises from cement shrinkage during curing and presents as pores randomly located along the stem. Interfacial gaps are much larger stem-cement separations caused by air introduced during stem insertion. To investigate the mechanical consequences of both types of defect, a finite element analysis model was created on the basis of a computed tomography image of a Charnley-Kerboul stem, and alternating torsional and transverse loads were applied. The propagation of fatigue cracks within the cement and the rotational stability of the stem were assessed in models simulating increasing amounts of interfacial gaps and pores. Anterior gaps covering at least 30 per cent of the implant surface promoted cement cracks and destabilized the stem. Anterolateral gaps were less destabilizing, but had more potential to promote cracks. In both cases, cracks occurred mainly outside gap regions, in areas where the stem contacted the cement during cyclic loading. Although random interfacial pores did not destabilize the implant, they acted as crack initiators even at low fractions (10 per cent). In conclusion, random interfacial pores were more harmful for the cement mantle integrity than were larger regions of interfacial gaps, although gaps were more detrimental for the rotational stability of the stem.
Asunto(s)
Artroplastia de Reemplazo de Cadera/instrumentación , Artroplastia de Reemplazo de Cadera/métodos , Cementos para Huesos/química , Prótesis de Cadera , Ensayo de Materiales , Modelos Biológicos , Falla de Prótesis , Adhesividad , Simulación por Computador , Análisis de Falla de Equipo , Análisis de Elementos Finitos , Humanos , Estrés MecánicoRESUMEN
This paper presents a tryptophan phosphorescence spectroscopy study on the membrane-bound mannitol transporter, EII(mtl), from E. coli. The protein contains four tryptophans at positions 30, 42, 109, and 117. Phosphorescence decays in buffer at 1 degrees C revealed large variations of the triplet lifetimes of the wild-type protein and four single-tryptophan-containing mutants. They ranged from <70 microseconds for the tryptophan at position 109 to 55 ms for the residue at position 30, attesting to widely different flexibilities of the tryptophan microenvironments. The decay of all tryptophans is multiexponential, reflecting multiple stable conformations of the protein. Both mannitol binding and enzyme phosphorylation had large effects on the triplet lifetimes. Mannitol binding induces a more ordered structure near the mannitol binding site, and the decay becomes significantly more homogeneous. In contrast, enzyme phosphorylation induces a large relaxation of the protein structure at the reporter sites. The implications of these structural changes on the coupling mechanism between the transport and the phosphorylation activity of EII(mtl) are discussed. Taken as a whole, our data show that tryptophan phosphorescence spectroscopy is a very sensitive technique to explore conformational dynamics in membrane proteins.
Asunto(s)
Proteínas Portadoras/química , Proteínas de la Membrana/química , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/química , Espectrometría de Fluorescencia/métodos , Triptófano/química , Sitios de Unión/genética , Transporte Biológico/genética , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cisteína/química , Cisteína/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli , Congelación , Vidrio , Histidina/química , Histidina/metabolismo , Mediciones Luminiscentes , Manitol/química , Manitol/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Monosacáridos , Mutagénesis Sitio-Dirigida , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Fosforilación , Soluciones , Termodinámica , Triptófano/genéticaRESUMEN
This paper presents a deceptively straightforward experimental approach to monitoring membrane protein-ligand interactions in vesicles and in living Escherichia coli cells. This is achieved via the biosynthetic incorporation of 7-azatryptophan, a tryptophan analogue with a red-shifted absorption spectrum, allowing collection of the emission signal of the target protein in a high tryptophan background via red-edge excitation. The approach is demonstrated for the mannitol permease of E. coli (EII(mtl)), an integral membrane protein of 637 amino acids, including four tryptophans, and single-tryptophan mutants of EII(mtl). By using a tryptophan auxotroph, a high level of 7-azatryptophan incorporation in EII(mtl) was achieved. The change in emission signal of the purified enzyme upon mannitol binding (-28%) was 4-fold larger than with EII(mtl) containing tryptophan, demonstrating the known higher sensitivity of this analogue for changes in the microenvironment [Schlesinger, R. (1968) J. Biol. Chem. 243, 3877-3883]. Changes in emission signal could also be monitored (-5%) when the enzyme was situated in vesicles, although it constituted only 10-15% of the total cytoplasmic membrane fraction. Of the five single-tryptophan mutants, the emission signal of the mutant with 7-azatryptophan at position 198 was the most sensitive for mannitol binding. Changes in emission signal not only were observed in vesicles (-18%) but also could be monitored in viable cells (-5%). The fact that only modest expression levels and no protein purification are needed makes this a useful approach for the characterization of numerous protein systems under in vitro and in vivo conditions.
Asunto(s)
Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Triptófano/análogos & derivados , Sitios de Unión , Membrana Celular/química , Membrana Celular/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Ligandos , Manitol/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Mutagénesis Sitio-Dirigida , Periplasma/química , Periplasma/metabolismo , Espectrometría de Fluorescencia , Triptófano/biosíntesis , Triptófano/genética , Triptófano/metabolismoAsunto(s)
Proteínas Bacterianas , Escherichia coli/enzimología , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/química , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/química , Sitios de Unión , Proteínas de Escherichia coli , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos , Termodinámica , Difracción de Rayos XRESUMEN
This paper reports that the aggregation state of a membrane protein can be changed reversibly without the use of chaotropic agents or denaturants by altering the attractive interactions between micelles of polyethylene glycol-based detergents. This has been documented using mannitol permease of Escherichia coli (EIImtl), a protein whose activity is dependent on the dimerization of its membrane-embedded domains. We show that the driving force for the hydrophobic interactions responsible for the dimerization can be decreased by bringing the protein into a less polar environment. This can be done simply and reversibly by increasing the micelle cluster size of the solubilizing detergent since the micropolarity in the micelle decreases upon clustering and is directly related to the cluster size. The micelle cluster size was varied at a fixed temperature by adding sodium phosphate or a second detergent with a distinct clustering behavior, and the changes were quantified by quasi-elastic light scattering and by determining the cloud point or demixing temperature (Td) of the detergent. Maximal EIImtl activity was found when no micelle clustering occurred, but the activity gradually decreased down to 5% of the maximal activity with increasing cluster size. The inactivation was found to be completely reversible. The kinetics of heterodimer formation were also significantly affected by changes in the micelle cluster size as expected. Increasing the cluster size resulted in faster formation of functional heterodimers by increasing the rate of homodimer dissociation. This phenomenon should be generally applicable to controlling the oligomeric state of membrane-bound proteins or even water-soluble proteins if their subunit association is dominated by hydrophobic forces.
Asunto(s)
Escherichia coli/química , Proteínas de la Membrana/metabolismo , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/química , Conformación Proteica/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Detergentes/farmacología , Dimerización , Proteínas de Escherichia coli , Cinética , Micelas , Proteínas de Transporte de Monosacáridos , Tamaño de la Partícula , Fosfoenolpiruvato/metabolismo , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Fosforilación , Polietilenglicoles/farmacología , Dispersión de RadiaciónRESUMEN
The relationship between vaginal temperature and ovulation time was studied in sows. The vaginal temperature was measured continuously between Day 4 and Day 10 after Altrenogest-treatment in 10 sows. Oestrus was checked with a vasectomized boar at 8-h intervals, and during oestrus, ovulation time was checked with transrectal ultrasonography at 2-h intervals between 07:00 h and 23:00 h. Two sows ovulated between 23:00 h and 07:00 h, and these sows were taken out of the experiment. In the eight remaining sows, a clear day/night rhythm in vaginal temperature was found: between 03:00 h and 09:00 h, vaginal temperature (LSM +/- sem, corrected for sow) was on average 38.2 +/- 0.01 degrees C; between 15:00 h and 21:00 h, vaginal temperature was on average 38.5 +/- 0.01 degrees C (P < 0.001). Between 4 days before ovulation and 2 days after ovulation, no changes in temperature could be found that were related to ovulation time in any of the sows. Therefore, in sows, changes in vaginal temperature cannot be used as a predictor for ovulation time, and consequently cannot be used to predict the best time for insemination.
Asunto(s)
Temperatura Corporal/fisiología , Detección del Estro/métodos , Ovulación/fisiología , Vagina/fisiología , Animales , Sincronización del Estro/efectos de los fármacos , Femenino , Periodicidad , Congéneres de la Progesterona/farmacología , Porcinos , Acetato de Trembolona/análogos & derivados , Acetato de Trembolona/farmacologíaRESUMEN
The effects of substrate and substrate analogue binding and phosphorylation on the conformational dynamics of the mannitol permease of Escherichia coli were investigated, using time-resolved fluorescence spectroscopy on mutants containing five single tryptophans situated in the membrane-embedded C domain of the enzyme [Swaving Dijkstra et al. (1996) Biochemistry 35, 6628-6634]. Since no fluorescent impurities are present in these mutants, the changes in fluorescence and anisotropy could be related with changes in the tryptophan microenvironment. Tryptophans at positions 30 and 42 showed changes in fluorescence intensity decay upon binding mannitol, which were reflected in the changes in lifetime distribution patterns. The disappearance of the shortest-lived decay component in these mutants, as well as in the mutant with a single tryptophan at position 109, indicates a change in the local environment such that quenching via neighboring side chains or solvent is reduced. Phosphorylation at histidine 554 and cysteine 384, located in the cytoplasmatic A and B domains of EII(mtl), respectively, induced an increase in the average fluorescence lifetimes of all of the tryptophans. The effect was most pronounced for tryptophans 30 and 109 which show large increases in the average fluorescence lifetime mainly due to loss of short-lived decay components. A correlation time distribution of the individual tryptophans deduced from an analysis of the anisotropy decay showed that they differed in their rotational mobility with tryptophan 30 showing the least local flexibility. Phosphorylation resulted in immobilization of W109 which, together with changes in the average fluorescence lifetime, is evidence for a conformational coupling between the phosphorylated B domain and the C domain. The influence of mannitol binding on the rotational behavior of the tryptophans is limited; it induces more internal flexibility at all tryptophan positions. A rotational correlation time of 30 ns was resolved for tryptophan 30, which probably represents a rotational mode of the micelle-embedded C-domain of EII(mtl) or a portion thereof. Upon phosphorylation, this rotational correlation time increases to 50 ns, probably reflecting a changed spatial orientation of W30 with respect to the C domain. Although kinetic experiments have shown that none of the tryptophans is essential for the catalytic activity of EII(mtl), it is significant that the residues most sensitive to mannitol binding, W30 and W42, are both located in the first membrane-spanning alpha-helix, a portion of which is highly conserved among mannitol-specific EII's of different bacteria.
Asunto(s)
Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/química , Triptófano/química , Detergentes , Proteínas de Escherichia coli , Polarización de Fluorescencia , Heptosas/metabolismo , Yoduros/metabolismo , Manitol/metabolismo , Micelas , Proteínas de Transporte de Monosacáridos , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/genética , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Fosforilación , Mutación Puntual , Unión Proteica , Espectrometría de Fluorescencia , Triptófano/genética , Triptófano/metabolismoRESUMEN
Investigation of the membrane-embedded mannitol permease of Escherichia coli (EIImtl) steady-state tryptophan fluorescence was hampered by fluorescent impurities arising from detergents and other sources during the isolation. The signals from these impurities could not be distinguished from tryptophan fluorescence on the basis of lifetimes or emission spectra. Consequently, a tryptophan-minus mutant of EIImtl, EIImtl(Trp-), was constructed to address this problem. The findings were that the fluorescent impurities, present in the detergents and/or arising from the action of the detergents on plastic vials and tubing used during the isolation procedure, accumulate in enzyme solutions to levels comparable to the signal from the tryptophan residues in the protein. The high affinity of these impurities for EIImtl makes them impossible to remove by dialysis, by reconstitution of the protein with pure phospholipids, or by detergent exchange when the protein is immobilized on a resin. A procedure was developed to completely remove all fluorescent impurities from the nonionic polyethylene glycol-based detergent, decylpenta(ethylene glycol) (C10E5). This detergent and modified isolation procedures yield EIImtl(Trp-) and single tryptophan mutants in which the impurities no longer interfere with the tryptophan emission signal. The methodologies presented in this paper might make it possible to study the fluorescence of tryptophan residues in other membrane proteins without the interference of impurities with similar fluorescence properties.
Asunto(s)
Detergentes , Proteínas de la Membrana/aislamiento & purificación , Contaminación de Medicamentos , Manitol/química , Proteínas de la Membrana/química , Plásmidos/metabolismo , Mutación Puntual , Espectrometría de Fluorescencia , TriptófanoRESUMEN
The fluorescence properties of six different single Trp mutants of the mannitol-specific transporter of Escherichia coli were studied in order to derive structural information at different locations in the enzyme. The use of pure detergent and special protein purification protocols was essential for reliable fluorescence spectra, as judged from tyrosine-like fluorescence in a tryptophan-minus mutant (Robillard et al., 1996). The steady-state fluorescence spectra of EIImtl mutants with single tryptophan residues at positions 30, 42, 109, 117, 320, and 384 provided information concerning the polarity of the environment and the effects of mannitol binding at these positions. Tryptophan positions 42, 109, and 117 with emission maxima ranging from 337 to 340 nm are relatively polar, and position 384 with an emission maximum at 346 nm is highly polar, whereas position 30 is highly apolar with a maximum at 324 nm. The fluorescence characteristics of tryptophan 30 suggest a buried position in a hydrophobic part of the enzyme, which is confirmed by the low Stern-Volmer quenching constant for I- quenching. Positions 109 and 117 show the highest quenching constants, indicating the most exposed positions, whereas positions 320 and 42 are moderately quenched, by I-. The tryptophan residue at position 384 is, even in the absence of externally added quencher, very strongly quenched, possibly by the carboxylate from aspartate 384 or by a tyrosinate at position 458 which is nearby in the folded protein (AB et al., in preparation; van Montfort et al., in preparation). The observed emission maxima and accessibilities of the tryptophans at the different positions are consistent with the predicted topology of the enzyme (Sugiyama et al., 1991). When mannitol is bound to wild-type EIImtl, an increase in fluorescence emission intensity was observed (Wood, 1988) which can now be attributed primarily to increased fluorescence intensity of the tryptophan at position 30.
Asunto(s)
Escherichia coli/enzimología , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/química , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Triptófano , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Membrana Celular/enzimología , Membrana Celular/ultraestructura , Cartilla de ADN , Proteínas de Escherichia coli , Cinética , Manitol/metabolismo , Modelos Estructurales , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos , Mutagénesis Sitio-Dirigida , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/aislamiento & purificación , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia/métodosRESUMEN
At present, inhaled glucocorticoids are widely accepted as the therapy of choice in chronic asthma. Treatment with inhaled glucocorticoids significantly suppresses local airway inflammation in asthmatics, but may also have systemic effects, e.g. a reduction of the number of circulating hypodense eosinophils or a down-modulation of HLA-DR antigen (Ag) expression by T lymphocytes in peripheral blood. However, the effect of long-term therapy with inhaled glucocorticoids on peripheral blood monocytes (PBM), which are the precursors of the most numerous cell type in the lung, the alveolar macrophage, have not yet been evaluated. We therefore investigated the expression of various cell surface Ag on PBM from non-smoking patients with allergic asthma who were treated for 2.5 years with a beta(2)-receptor agonist plus either an inhaled glucocorticoid (beclomethasone dipropionate, BDP) (n = 4) or an anticholinergic or placebo (n = 8). We compared the results with healthy volunteers (n = 7). Long-term treatment of allergic asthmatics with inhaled BDP, but not anticholinergic or placebo therapy, was associated with a significantly lower CDllb Ag expression (p < 0.04) and higher expression of CD13, CD14 and CD18 Ag (p < 0.05, p < 0.02 and p < 0.04, respectively) when compared with the healthy control subjects (n = 7). Most interestingly, PBM of asthmatics treated with inhaled BDP expressed an almost two-fold higher level of CD14 Ag on their cell surface than PBM of patients treated with anticholinergic or placebo (p < 0.03). No significant differences in the expression of CD16, CD23, CD25, CD32 and CD64 Ag or HLA-DR were observed between PBM from the different patient groups or healthy controls. Taken together, this study shows that long-term local therapy with inhaled BDP coincides with an altered expression of at least one cell surface Ag on PBM from allergic asthmatics.
RESUMEN
It is easily forgotten that not yet a hundred years ago the only way to look into the patients' body was via invasive procedures. Within the year of the discovery of X-rays by Conrad Röntgen the need for three dimensional imaging had been voiced. The driving force behind this development was undoubtedly clinical motivation. Planar X-radiographs were not satisfactory to the clinicians who urged the radiologists to provide them with tomographic images. Between 1910 and 1940, classical tomography has been the product of individuals rather than collective groups. It is only in the mid thirties that scientists found out about each other and started to correspond vigorously. Mayer was the first to suggest in 1914 the idea of tomography. Bocage, Grossman and Vallebona all developed the idea further and built their own equipment. In 1931 Ziedses des Plantes published the most extensive and thorough study on tomography. In the forties and fifties a stagnation is noticed, only further refinements to the existing equipment are carried out. Although Frank and Takahashi published the basic principles of axial tomography in the mid forties, we had to wait for the necessary developments in electronics before Hounsfield was able to develop and commercialize the first axial computer tomography in 1972 (EMI-Scanner). At the time all the big radiology companies rushed into the field and soon, second, third and fourth generation CT scanners became available. Only a few years later a new way of generating images without using ionizing radiation was introduced. Lauterbur and Damadian produced the first low quality images with magnetic resonance, a technique called zeugmatography by its inventors. In 1974 the first images of a living subject were published and initial scepticism was replaced by euphoria. This resulted in the spectacular evolution in Magnetic Resonance that we are now observing. While it is impossible to predict the future, the development of networks, the increase in data acquisition and storage will spread a new light on our specialty. A closer cooperation between radiologists, pathologists and clinicians will undoubtedly be necessary, as well as a partial redefinition of the radiologists task.
Asunto(s)
Tomografía por Rayos X/historia , Historia del Siglo XX , Humanos , Imagen por Resonancia Magnética/historia , Tomografía Computarizada por Rayos X/historiaRESUMEN
IL-4 up-regulates various monocytic properties that are associated with pro-inflammatory functions. Paradoxically, IL-4 may also act as an anti-inflammatory agent by down-regulating the production of several inflammatory mediators. As the activity of some mediators has recently been shown to be regulated by peptidases, we examined whether IL-4 was able to modulate the expression of a cell membrane-associated peptidase, aminopeptidase-N (CD13). IL-4 caused a dose-dependent increase in the expression of CD13 Ag on highly purified human blood monocytes. Maximal expression was observed around 48 h of culture. This IL-4-induced increase was completely blocked by anti-IL-4 antiserum. Furthermore, the increase in surface expression was preceded by increased mRNA levels of CD13, which was maximal around 24 h of culture. We also observed that CD13-mediated leucine-aminopeptidase activity of monocytes was induced by IL-4. Other CD13-expressing cells were also sensitive to IL-4, as CD13 Ag expression and CD13 mRNA levels were up-regulated in human alveolar macrophages and endothelial cells upon IL-4 treatment. The increased expression of cell membrane aminopeptidase-N represents a potentially increased cellular ability to inactivate inflammatory mediators. Therefore, these findings represent further evidence of IL-4-mediated anti-inflammatory actions. We postulate that up-regulation of aminopeptidase-N expression may be an indirect mechanism of IL-4 to modulate the action of bioactive peptides. This mechanism may underlie, at least partially, the anti-inflammatory effects of IL-4 in vivo.
Asunto(s)
Aminopeptidasas/biosíntesis , Antígenos CD/biosíntesis , Antígenos de Diferenciación Mielomonocítica/biosíntesis , Endotelio Vascular/enzimología , Interleucina-4/fisiología , Macrófagos Alveolares/enzimología , Monocitos/enzimología , Aminopeptidasas/efectos de los fármacos , Northern Blotting , Antígenos CD13 , Línea Celular , Células Cultivadas , Citocinas/fisiología , Endotelio Vascular/citología , Humanos , Leucil Aminopeptidasa/metabolismo , ARN Mensajero/biosíntesis , Regulación hacia ArribaRESUMEN
IL-4 has multiple biologic activities and it has been shown to have effects on B and T lymphocytes, mast cells, NK cells, and monocytes. We studied the influence of IL-4 on the expression of cell membrane determinants, in particular aminopeptidase-N (CD13) and Fc epsilon RIIb (CD23), on human peripheral blood monocytes. We compared the response of monocytes with the response of human alveolar macrophages and monocytic cell lines (U937 and THP1), as mature and more immature representatives of the mononuclear phagocyte system, respectively. A dose-dependent increase of the expression of CD13 Ag was observed when monocytes were cultured with IL-4. Kinetic analyses revealed that this induction was maximal after 2 to 3 days of culture and resembled the kinetics of IL-4-induced expression of Fc epsilon RIIb on monocytes. This IL-4-induced increase was absent when monocytes were cultured with IL-4 and an anti-IL-4 antiserum. Concomitantly, an IL-4-induced increase in leucine-aminopeptidase activity could be observed. Northern blot analysis showed that incubation of monocytes with IL-4 induced a marked increase in CD13 mRNA. Alveolar macrophages also exhibited an increase in CD13 Ag expression when exposed to IL-4. Surprisingly, IL-4 was unable to induce expression of Fc epsilon RIIb on alveolar macrophages. U937 and THP1 cells did not show an induction of CD13 Ag when cultured in the presence of IL-4. However, IL-4 did induce the expression of Fc epsilon RIIb on both cell lines, suggesting the presence of functional IL-4R. Our data demonstrate that IL-4 increases the expression of CD13 Ag on monocytes. This IL-4-induced increase can also be observed in more mature monocytic cells such as alveolar macrophages, but is absent in immature cells such as U937 or THP1 cells. This is functionally accompanied by an increase in leucine-aminopeptidase activity and may be part of the general activation of monocytes/macrophages by IL-4. In conclusion, the data suggest that IL-4 responsiveness, in particular the induction of CD13 Ag and Fc epsilon RIIb expression, may be dependent on the stage of maturation of monocytes/macrophages.
Asunto(s)
Aminopeptidasas/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Interleucina-4/farmacología , Macrófagos/metabolismo , Monocitos/metabolismo , Receptores Fc/metabolismo , Aminopeptidasas/genética , Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/genética , Secuencia de Bases , Antígenos CD13 , Diferenciación Celular , Células Cultivadas , Expresión Génica/efectos de los fármacos , Humanos , Técnicas In Vitro , Receptores de Lipopolisacáridos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Datos de Secuencia Molecular , Monocitos/citología , Monocitos/efectos de los fármacos , Oligodesoxirribonucleótidos/química , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Receptores de IgE , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
The gene encoding the major vitellogenin from chicken has been completely sequenced and its exon-intron organization has been established. The gene is 20,342 base-pairs long and contains 35 exons with a combined length of 5787 base-pairs. They encode the 1850-amino acid pre-peptide of vitellogenin, which is the precursor of the mature yolk proteins, the serine-rich and heavily phosphorylated phosvitin and the lipovitellin. The 217-amino acid phosvitin polypeptide occupies an internal position (residue 1112 through 1328) within the vitellogenin molecule. The 125,000 and 30,000 Mr lipovitellin polypeptides are encoded by the sequences at the N-terminal and the C-terminal sides of the phosvitin section, respectively. The main features of the gene and protein sequences, and the evolutionary implications, are discussed.
Asunto(s)
Pollos/genética , Proteínas del Huevo/genética , Vitelogeninas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN , Yema de Huevo , Exones , Genes , Intrones , Datos de Secuencia MolecularRESUMEN
Snapping turtle (Chelydra serpentina) ribonuclease was isolated from pancreatic tissue. Turtle ribonuclease binds much more weakly to the affinity chromatography matrix used than mammalian ribonucleases. The amino acid sequence was determined from overlapping peptides obtained from three different digests. The N-terminal amino acid sequence of the protein determined by others [E. A. Barnard, M. S. Cohen, M. H. Gold J.-K. Kim (1972) Nature (Lond.) 240, 395-398] and homology were used as additional evidence for several overlaps. The polypeptide chain consists of 119 amino acid residues. Compared to most ribonucleases the N-terminal residue, three residues in the loop near residue 71 and two residues in the loop near residue 114 are deleted, and there is one additional residue in the loop near residue 23. The half-cystines at positions 65 and 72, which form a disulfide bond in mammalian ribonucleases, are not present in turtle ribonuclease. Turtle ribonuclease differs from bovine ribonuclease at 70 of the 118 positions where both proteins have amino acid residues. Turtle ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Ala-Ser (positions 76-78).