RESUMO
A novel method for the direct measurement of the elusive magnetic and electric dipole moments of the τ lepton is presented. The experimental approach relies on the production of τ^{+} leptons from D_{s}^{+}âτ^{+}ν_{τ} decays, originating in fixed-target collisions at the LHC. A sample of polarized τ^{+} leptons is kinematically selected and subsequently channeled in a bent crystal. The magnetic and electric dipole moments of the τ^{+} lepton are measured by determining the rotation of the spin-polarization vector induced by the intense electromagnetic field between crystal atomic planes. The experimental technique is discussed along with the expected sensitivities.
RESUMO
Vertebral fractures occur in over 60% of newly diagnosed multiple myeloma (MM) patients and can cause pain, disability and poor quality of life. Antimyeloma therapy can lead to symptoms improvement, but these effects can take time to be perceived. Application of radiotherapy prior to peripheral blood stem cells (PBSC) mobilisation can impair stem cell collection. Percutaneous vertebroplasty has been proposed as a suitable option to rapidly relieve bone pain from vertebral fractures in MM patients, but, little is known about the effects of this procedure on subsequent PBSC mobilisation, collection and transplant. Eighteen patients (10M/8F, median age 64.5 years) with untreated MM and painful vertebral lesions underwent vertebroplasty prior to proceed to the planned transplant program at our Institution. Forty-one procedures were performed at C2-L5 levels, eight patients were treated at ≥2 levels. Ninety-five per cent of the cases obtained a complete or optimal pain control. All the patients successfully mobilised PBSC (median CD34+ cells = 10.8 × 10(6) /kg) and underwent autologous PBSC transplant; both polymorphonucleates and platelets recovery averaged 11 days. Our data seem to suggest that percutaneous vertebroplasty is useful in newly diagnosed MM patients with painful vertebral fractures as it allows rapid and durable achievement of pain control, without interfering with further treatment.
Assuntos
Fraturas por Compressão/cirurgia , Mobilização de Células-Tronco Hematopoéticas , Mieloma Múltiplo , Dor/prevenção & controle , Transplante de Células-Tronco de Sangue Periférico , Fraturas da Coluna Vertebral/cirurgia , Vertebroplastia/métodos , Adulto , Idoso , Feminino , Fraturas por Compressão/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/complicações , Mieloma Múltiplo/terapia , Medição da Dor , Qualidade de Vida , Fraturas da Coluna Vertebral/etiologiaRESUMO
In previous studies we identified a surface molecule (termed GL183) capable of mediating cell activation and selectively expressed by a subset of human CD3-CD16+ natural killer (NK) cells. In this study we analyzed whether other subset-specific functional molecules were expressed in GL183- NK cells. To this end, mice were immunized with the PE29 (CD3-CD16+GL183-) NK clone. Monoclonal antibodies (mAbs) were selected by screening the hybridoma supernatants for their ability to trigger the cytolytic activity of clone PE29 against the human myelomonocytic leukemia U937. The EB6 mAb (IgG1) triggered the PE29 clone, but not a GL183+ clone used as a control. EB6+ cells ranged between 1 and 13% of peripheral blood lymphocytes and were largely included in the CD3-CD16+CD56+ cell populations (only less than 2% of EB6+ cells were CD3+). Analysis of resting or activated CD3-CD16+ populations, or clones for the expression of EB6 or GL183 mAbs, allowed us to identify four distinct, phenotypically stable, NK subsets (EB6+GL183-; EB6+GL183+; EB6-GL183+; EB6-GL183-). Similar to GL183 mAb, the EB6 mAb selectively triggered the NK subset expressing the corresponding surface antigen to lyse human tumor cell lines including U937, IGROV-I, M14, and A549. In addition, EB6 mAb sharply inhibited the cytolytic activity of EB6+ clones against P815, M12, and P3U1 murine target cells. In EB6+GL183+ ("double-positive") clones both EB6 and GL183 mAb inhibited the redirected killing of P815 cells induced by anti-CD16, anti-CD2 mAbs and phytohemagglutinin (PHA). Similar to GL183 molecules, molecules precipitated by EB6 mAb were represented by either single 58-kD chain or double chains of 55 and 58 kD (with no detectable differences in EB6+GL183- or EB6+GL183+ clones). In sequential immunoprecipitation experiments using the double-positive clones CEG52 and CA25.50, preclearing of cell lysates with EB6 or GL183 mAb removed only EB6 or GL183 molecules, respectively, thus indicating that the two antigenic determinants are carried by two distinct molecules. Peptide map analysis indicated that EB6 (or GL183) molecules precipitated from double-positive clones were identical to the corresponding molecules isolated from single-positive ones. On the other hand, comparison of the EB6 and GL183 maps revealed peptides that were unique to each molecule, although most of the major peptides migrated to identical positions. We further investigated whether correlation existed between the phenotypic assignment of NK clones and their ability to mediate specific lysis of normal allogeneic cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação/imunologia , Antígenos de Superfície/imunologia , Isoantígenos/imunologia , Células Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores Fc/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Anticorpos Monoclonais , Antígenos de Superfície/isolamento & purificação , Western Blotting , Complexo CD3 , Células Cultivadas , Células Clonais , Citotoxicidade Imunológica , Eletroforese em Gel de Poliacrilamida , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Peso Molecular , Receptores de IgGRESUMO
The GL183 mAb was obtained by immunizing BALB/c mice with the E57 clone (CD7+CD2+CD3-CD16+CD56+) derived from human peripheral blood NK cells. In human peripheral blood, GL183-reactive cells ranged between 2 and 12% (mean 6.5%) in 10 different donors. Double fluorescence and FACS analysis showed that GL183+ cells were consistently included in the CD56+ or CD16+ cell populations. Moreover, since only a fraction of CD56+ or CD16+ cells (approximately 40%) coexpressed GL183 surface antigen, reactivity with GL183 mAb appears to define two subsets within the CD3- lymphocyte population expressing NK cell markers. Although, the majority of GL183+ cells were CD3-, approximately 1% expressed CD3 surface antigens. As shown by clonal analysis, these infrequent CD3+GL183+ cells coexpressed CD56 and CD16 antigens. Cloning of CD3-GL183+ or CD3-GL183- cell populations under limiting dilution conditions yielded clonal progenies that maintained their original surface phenotype. Therefore, expression or lack of expression of GL183 surface antigens represents a stable phenotypic property of a subset of human CD3- NK cells. Immunoprecipitation experiments and two-dimensional PAGE analysis indicated that GL183-reactive molecules were represented in different clones either by a single 58-kD chain or, more frequently, by two chains of approximately 55 and approximately 58 kD, respectively. Analysis of GL183+ or GL183- NK clones for their ability to lyse human (IGROV I) or murine (P815) tumor target cells indicated that GL183- clones were, on average, fivefold more efficient in inducing target cell lysis. GL183+ and GL183- clones produced comparable levels of TNF-alpha in response to PHA plus PMA or anti-CD16 mAb plus PMA. Importantly, production of TNF-alpha was also induced by stimulation of GL183+ clones with GL183 mAb plus PMA. These data indicated that GL183 antigen could mediate cell triggering. This concept was confirmed by the analysis of Ca2+ mobilization, as GL183 mAb induced (in GL183+ clones) increments of [Ca2+]i comparable with those induced by PHA. Moreover, GL183 mAb, or its F(ab')2 fragments, strongly enhanced the cytolytic activity of GL183+ clones against a panel of human tumor target cells, including U937, Raji, IGROV I, M14, and A549. In contrast, GL183 mAb, but not the F(ab')2 fragments, sharply inhibited the cytolytic activity of the same clones against P815, M12, and P3U1 murine target cells. In this case, the effect of GL183 mAb (inhibition) was opposite that of PHA or of stimulatory anti-CD2 or anti-CD16 mAbs, which consistently enhanced the target cell lysis.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos de Diferenciação/análise , Antígenos de Superfície/análise , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/análise , Receptores Fc/análise , Animais , Anticorpos Monoclonais , Antígenos de Diferenciação/imunologia , Antígenos de Superfície/fisiologia , Complexo CD3 , Cálcio/metabolismo , Células Clonais , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Receptores Fc/imunologia , Receptores de IgG , Fator de Necrose Tumoral alfa/biossínteseRESUMO
We propose a unique program of measurements of electric and magnetic dipole moments of charm, beauty and strange charged baryons at the LHC, based on the phenomenon of spin precession of channeled particles in bent crystals. Studies of crystal channeling and spin precession of positively- and negatively-charged particles are presented, along with feasibility studies and expected sensitivities for the proposed experiment using a layout based on the LHCb detector.
RESUMO
Cytoplasmic monomeric hemoglobin I from the bacteria-harboring gill of the bivalve mollusc Lucina pectinata has been crystallized in a form suitable for atomic resolution X-ray structural investigations. The crystals have been grown at pH 4.8, in 0.05 M-acetate buffer, using 2.6 M-ammonium sulfate as precipitating agent. The crystals belong to the monoclinic space group P2(1), with unit cell constants a = 50.0 A, b = 38.6 A, c = 42.1 A, beta = 107.1 degrees, and contain one molecule (14,000 Mr) in the asymmetric unit. By means of single crystal microspectrophotometry it has been shown that the crystals contain the ferric form of L. pectinata "sulfide reactive" hemoglobin I. On the other hand, by careful control of the buffering medium composition, it has been possible to obtain stable crystals of the deoxy, oxy and sulfide forms of the protein.
Assuntos
Bivalves/química , Cristalografia , Compostos Férricos/química , Hemoglobinas/química , AnimaisRESUMO
The rate of catalyzed hydrolysis of the acyl enzyme analogs indolacryloyl-gamma-chymotrypsin and furylacryloyl-gamma-chymotrypsin in the crystal has been measured by single crystal microspectrophotometry and compared with the rate of catalyzed hydrolysis of acyl-gamma-chymotrypsin in solution and acyl-alpha-chymotrypsin both in solution and in the crystal. The maximal deacylation rate is the same for both species and independent of the physical state. However, the pH dependence of the deacylation rate of crystalline acyl-gamma-chymotrypsin shows a 0.9 unit shift in the pK of the catalytic system which is unique and probably consequent to specific lattice interactions.
Assuntos
Quimotripsina/metabolismo , Acilação , Cristalização , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , SoluçõesRESUMO
The intrathecal production of antimeasles antibodies was studied using the enzyme-linked immunosorbent assay in eight specimens of serum and cerebrospinal fluid (CSF) from patients with clinical signs of subacute sclerosing panencephalitis (SSPE). The test was performed using a 1:5 dilution of CSF and a 1:2000 dilution of serum (ratio 1:400) in order to nullify the physiological gradient of immunoglobulins across the blood brain barrier (BBB). This procedure allowed a rapid and accurate assessment of the synthesis of specific immunoglobulins in the CSF and a good evaluation of the permeability of the BBB. A diagnosis of SSPE was provided in five out of eight patients with clinical signs of the disease. Clinical follow-up confirmed the diagnosis of SSPE in the group of patients with clear evidence of intrathecal synthesis of antimeasles antibodies.
Assuntos
Anticorpos Antivirais/líquido cefalorraquidiano , Imunoglobulina G/líquido cefalorraquidiano , Imunoglobulina M/líquido cefalorraquidiano , Vírus do Sarampo/imunologia , Panencefalite Esclerosante Subaguda/líquido cefalorraquidiano , Adolescente , Adulto , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Barreira Hematoencefálica , Criança , Citomegalovirus/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Herpesvirus Humano 3/imunologia , Humanos , Masculino , Vírus da Caxumba/imunologia , Vírus da Rubéola/imunologia , Testes Sorológicos , Simplexvirus/imunologia , Panencefalite Esclerosante Subaguda/imunologiaRESUMO
In the newborn and foetus in the last months of gestation it is possible to see in lateral radiography of the spine, a longitudinal radiotransparent band which may divide the bone nucleus of the vertebral body into two portions. This front vertebral separation should be considered an expression of a reversible abnormality rather than a malformation to the extent that it almost always concerns a differentiation delay in the vertebral structures. The literature and personal cases show, in fact, that the finding disappears more or less rapidly.
Assuntos
Doenças do Recém-Nascido , Coluna Vertebral/anormalidades , Diagnóstico Diferencial , Humanos , Recém-Nascido , Doenças do Recém-Nascido/diagnóstico , Disrafismo Espinal/diagnósticoRESUMO
Monoclonal antibodies (MoAbs) specific for surface molecules involved in lymphocyte activation, represent an useful tool for the analysis of the activation pathways of different effector lymphocytes. We have analyzed the ability of MoAbs directed against "triggering" surface molecules, such as CD3/TCR, CD2 and CD16 to induce activation of the lytic machinery in T and NK lymphocytes, using a redirected killing assay. In addition, we have constructed bispecific monoclonal antibodies (BiMoAbs) directed against both the CD3/TCR complex (or the CD16 molecule) and a tumor associated antigen expressed on the surface of ovarian cancer cells. BiMoAbs were constructed by two different approaches: a) conjugation of two distinct MoAbs by a chemical heterobifunctional agent; b) selection of hybrid-hybridomas secreting BiMoAbs. BiMoAbs were able to focus appropriate lymphoid effector cells on tumor cells expressing the relevant antigen, and to induce tumor cell lysis. Therefore, these reagents were able to confer a new specificity for a given antigen to effector lymphocytes, independently from the original one. For these properties, BiMoAbs may represent a suitable tool for new strategies of adoptive immunotherapy, based on the use of effector cells that have been specifically armed by BiMoAbs against the tumor.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Especificidade de Anticorpos , Antígenos de Neoplasias/imunologia , Complexo CD3 , Humanos , Hibridomas/efeitos dos fármacos , Hibridomas/enzimologia , Hibridomas/imunologia , Células Matadoras Naturais/imunologia , Seleção Genética , Linfócitos T/imunologiaRESUMO
The classification, pathogenesis, clinical features and treatment of pemphigus are examined with emphasis' on the histopathological similarities of the elementary intraepithelial lesion and the histotopographical differences at epithelial level that differentiate the various types of pemphigus. In a detailed review of the pathogenesis of the condition, the circumstances propitious for the onset of pemphigus are identified as an HLA phenotype and the autoantigenic denaturation of the glycocalyx. The condition is described as vesicolobullous and is differentiated from similar bullous and non-bullous dermatoses. The most significant pictures of oral pemphigus are specified as are the basic elements of general and local treatment.
Assuntos
Doenças Autoimunes/imunologia , Doenças da Boca/etiologia , Pênfigo , HumanosRESUMO
The Authors give a detailed analysis of the potential virulence factors and possible pathogenic processes associated with candida. They qualify the interactions between local, general, natural and acquired defences, setting out the possible deficiency involved in the emergence of secondary candidiasis. They make a classificatory revision of forms of candidiasis on the basis of topographic, anatomo-pathologic criteria and of the course of the disease. They investigate the most significant aspects of the situations described especially in oral localization. An evaluation is made of chronic mucocutaneous candidiasis as a pathological affection in itself and in its correlation with H.I.V. infection.
Assuntos
Candida , Candidíase Bucal , Síndrome da Imunodeficiência Adquirida/complicações , Candida/imunologia , Candida/patogenicidade , Candida/fisiologia , Candidíase Bucal/classificação , Candidíase Bucal/etiologia , Candidíase Bucal/imunologia , Candidíase Bucal/microbiologia , Humanos , ImunidadeRESUMO
Autologous stem cell transplantation is considered the standard of care for multiple myeloma patients aged < 65 years with no relevant comorbidities. The addition of drugs acting both on bone marrow microenvironment and on neoplastic plasma cells has significantly increased the proportion of patients achieving a complete remission after induction therapy, and these results are mantained after high-dose melphalan, leading to a prolonged disease control. Studies are being carried out in order to evaluate whether short term consolidation or long-term maintenance therapy can result into disease eradication at the molecular level thus increasing also patients survival. The efficacy of these new drugs has raised the issue of deferring the transplant after achiving a second response upon relapse. Another controversial point is the optimal treatment strategy for high-risk patients, that do not benefit from autologous stem cell transplantation and for whom the efficacy of new drugs is still matter of debate.
Assuntos
Hemeproteínas , Imidazóis , Metamioglobina , Animais , Aplysia , Sítios de Ligação , Ligantes , Conformação Proteica , Espectrofotometria , Baleias , Difração de Raios XRESUMO
We demonstrate the photoassociation of ultracold rubidium dimers using coherent femtosecond pulses. Starting from a cloud of ultracold rubidium atoms, electronically excited rubidium molecules are formed with shaped photoassociation pump pulses. The excited state molecules are projected with a time-delayed probe pulse onto molecular ion states which are detected in a mass spectrometer. Coherent transient oscillations of the excited state population are observed in the wings of the pump pulse, in agreement with the time-dependent solution of the Schrödinger equation of the excitation process.
RESUMO
The aim of this study was to analyze at the clonal level the phenotype and functions of T cells from patients with severe aplastic anemia (SAA). For this purpose we studied 175 T-cell clones obtained from peripheral blood (PB) and bone marrow (BM) of four SAA patients and 97 clones from two healthy controls. The percentage of CD8+ T-cell clones obtained from the patients' PB and BM was higher, but not significantly (P = .07 and P = .14, respectively), than that obtained in controls. A higher proportion of T-cell clones from SAA patients exhibited lectin-dependent cytolytic activity and especially natural killer-like activity when compared with controls (PB: P less than .01, P less than .05; BM: P less than .05, P less than .01, respectively). Lymphokine release was tested before and after mitogen stimulation. A number of patients' clones were able to release interferons (IFNs) spontaneously (PB: 28.6% v 0%, P less than .05; BM: 28.6% v 0%, P less than .10). After mitogen stimulation, patients' BM T-cell clones produced IFNs in greater proportions (90.9% v 46.7%, P less than .01) and in greater quantities (PB: 25.5 arbitrary units [AU]/mL v 5.7 AU/mL, P less than .03; BM: 26 AU/mL v 9.1 AU/mL, P = .011) as compared with controls. Tumor necrosis factor (TNF) activity was not found in supernatants of unprimed T-cell clones. After mitogen stimulation, PB T-cell microcultures produced TNF alpha in greater proportions (97.9% v 72.2%, P less than .01) and, also in this case, in greater quantities (PB: 7.2 AU/mL v 1.5 AU/mL, P = .007; BM: 9.9 AU/mL v 1.5 AU/mL, P = .003) than controls. In conclusion, T-cell clones from SAA patients exhibit predominantly a CD8+ phenotype, a greater cytotoxic activity, and can be shown to produce greater quantities of suppressor lymphokines when compared with controls.
Assuntos
Anemia Aplástica/sangue , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T Reguladores/fisiologia , Adolescente , Adulto , Antígenos de Diferenciação de Linfócitos T/análise , Medula Óssea/patologia , Complexo CD3 , Antígenos CD4/análise , Linfócitos T CD4-Positivos/imunologia , Antígenos CD8 , Separação Celular , Células Clonais , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Humanos , Imunofenotipagem , Interferons/biossíntese , Interleucina-3/biossíntese , Masculino , Glicoproteínas de Membrana/análise , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T/análise , Linfócitos T Reguladores/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
An enzyme-linked immunosorbent assay (ELISA) for the evaluation of growth kinetics of Toxoplasma gondii in tissue cultures was developed. Tissue culture microplates (96 wells) were seeded with Vero cells, infected with a virulent strain of T. gondii, and incubated for different time periods. The ELISA was performed with anti-T. gondii antibodies on the infected cells. The method was simple, rapid, and accurate, and very good correlations between the ELISA results and the percentage of T. gondii rosettes in infected cells, the number of free T. gondii in the supernatant, and the amount of T. gondii antigen in the supernatant were observed.
Assuntos
Antígenos de Protozoários/análise , Ensaio de Imunoadsorção Enzimática , Técnicas Imunoenzimáticas , Toxoplasma/crescimento & desenvolvimento , Animais , Células Cultivadas , Chlorocebus aethiops , Rim , Cinética , Fatores de Tempo , Toxoplasma/imunologiaRESUMO
Complex II (succinate-coenzyme Q reductase) was resolved into ten different polypeptides by polyacrylamide gel electrophoresis. Four polypeptides, CII-1, CII-2, CII-3, and CII-4 with molecular weights of 70 000, 24 000, 13 500, and 7000, were present in large amounts in all preparations examined. CII-1 and CII-2 are the flavoprotein and iron-sulfur protein, respectively, of succinate dehydrogenase; CII-3 and CII-4 have not been functionally indentified. Six polypeptides were present in much smaller amoumts as judged by staining intensity, and each of these comigrated with components in complex III. The amino acid compositions of several of the minor components in complex II were identical with that of an equivalently migrating polypeptide in complex III. We conclude that succinate-coenzyme Q reductase contains four different polypeptides and is contaminated with variable amounts of complex III when isolated as complex II.
Assuntos
Complexos Multienzimáticos , Oxirredutases , Peptídeos , Succinato Desidrogenase , Aminoácidos/análise , Animais , Bovinos , Mitocôndrias Cardíacas/enzimologia , Peso Molecular , UbiquinonaRESUMO
The analogue of ATP, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), binds tightly to pig muscle 3-phosphoglycerate kinase. A dissociation constant Kd of 0.0095 +/- 0.0015 mM was determined by fluorimetric titration on the basis of 1:1 stoichiometry. TNP-ATP is a strong competitive inhibitor towards MgATP and MgADP with a Ki of 0.008 +/- 0.001 mM for both substrates. It is also a mixed-type inhibitor towards 3-phosphoglycerate with similar inhibition constants. Binding of TNP-ATP to 3-phosphoglycerate kinase is accompanied by a tenfold intensity increase and a blue shift of about 20 nm in its fluorescence emission spectrum and a shift of the pK of its trinitrophenyl group towards a more acidic pH. These findings suggest that the negatively charged trinitrophenyl group of TNP-ATP significantly contributes to the binding of the analogue. By stepwise replacement of the fluorescent TNP-ATP, the dissociation constants (Kd) for ADP and MgADP binding were determined and found to be 0.78 +/- 0.08 and 0.048 +/- 0.006 mM respectively, which are consistent with the values previously determined by equilibrium dialysis [Molnár and Vas (1993) Biochem J. 293, 595-599]. In similar competitive-titration experiments, ATP and MgATP did not completely substitute for TNP-ATP. For the fraction of the analogue that could be substituted, the dissociation constants for MgATP and ATP were estimated to be 0.27 +/- 0.09 and 0.33 +/- 0.15 mM respectively, close to the values determined by equilibrium dialysis. Using the same method, a significant weakening of binding of both (Mg)ADP and (Mg)ATP could be detected in the presence of 3-phosphoglycerate: their respective Kd values became 0.34 +/- 0.04 and 0.51 +/- 0.22 mM. The reciprocal effect, i.e. weakening of 3-phosphoglycerate binding in the presence of the nucleotide substrates, has been observed previously [Vas and Batke (1984) Eur. J. Biochem. 139, 115-123]. Similarly, a much weaker binding of (Mg)ATP could be observed in the presence of 1,3-bisphosphoglycerate (Kd = 2.30 +/- 0.68 mM). The possible reason for the mutual weakening of substrate binding is discussed in the light of the available structural data.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Corantes Fluorescentes , Fosfoglicerato Quinase/antagonistas & inibidores , Fosfoglicerato Quinase/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Cristalografia por Raios X , Ácidos Difosfoglicéricos/farmacologia , Ácidos Glicéricos/farmacologia , Concentração de Íons de Hidrogênio , Músculos/enzimologia , Fosfoglicerato Quinase/química , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Espectrofotometria , SuínosRESUMO
Human thymocytes lacking both CD4 and CD8 differentiation antigens were prepared by treating total thymocyte suspensions with a mixture of anti-CD4 and anti-CD8 monoclonal antibodies and complement. The resulting populations contained less than 2% CD4+, CD8+ or WT31+ cells and variable percentages (less than 20%) of CD3+ cells. These cell populations were cultured in recombinant IL-2 in the presence of peripheral blood mononuclear cells as feeder cells. Cells underwent extensive proliferation accompanied by a progressive increase of CD3+ and CD8+ cells. On the other hand, appearance of neither WT31+, alpha/beta-positive T cell receptor (TCR), nor CD4+ cells could be observed in several independent experiments. Functional analyses revealed the appearance and the progressive increase of cytolytic activity against the natural killer (NK)-sensitive K562 cells as well as the NK-resistant fresh melanoma cells. Experiments of T cell cloning indicated that both the expression of CD8 and CD3 antigens and the appearance of cytolytic activity were consequent to cell maturation occurring at the level of CD4-CD8- non-cytolytic cell precursors. In these experiments, more than 30% of cells underwent clonal expansion and all the clonal progenies obtained displayed cytolytic activity and expressed the CD3+WT31- surface phenotype. The expression of CD8 was variable, whereas no CD4+ clones could be obtained. Cells expressing such surface phenotype are known to belong to the TCR gamma-positive T lymphocyte subset lacking the typical alpha/beta TCR and thus appear to be the only T cell type capable of in vitro proliferation and maturation under easily reproducible culture conditions.