The incubation of human peripheral blood lymphocytes (PBL) with the natural killer (NK)-sensitive MOLT-4 cell line results in PBL-target cell conjugate formation by certain lymphocyte subpopulations. Following velocity sedimentation, the PBL depleted of these conjugate-forming subpopulations are markedly diminished in the ability to mediate either antibody-dependent cellular cytotoxicity (ADCC) or NK activity. The immediate testing of highly pure PBL subpopulations isolated from the NK target conjugates does not reveal the expected recovery of augmented ADCC or NK levels. Following in vitro incubation, however, the PBL NK target-binding subpopulations do manifest augmented levels of both NK and ADCC, whereas the depleted PBL continue to display diminished NK and ADCC levels. In addition, the degree of augmented NK and ADCC levels recovered by the NK target-binding PBL subpopulations appears dependent on both the time and the temperature of in vitro incubation. Moreover, the ADCC recovery patterns are identical to those observed for NK activity regardless of the time and temperature of in vitro incubation. These results directly demonstrate that the PBL subpopulations isolated from certain NK target cells are functionally enriched in the ability to mediate from ADCC and NK activity. 相似文献
Adenylate cyclase (ATP pyrophosphate-lyase, EC 4.6.1.1) in plasma membranes from human thyroid was highly responsive to thyrotropin. Pretreatment of thyroid plasma membranes with 5′-guanylylimidodiphosphate (Gpp(NH)p) in the presence of Mg2+ led to a temperature-dependent activation, which was seen neither in the absence of Mg2+ nor at 4 °C. By contrast, thyrotropin bound to its receptors regardless of the temperature and produced its maximal effect after 2 min of preincubation in the absence or presence of Mg2+. Furthermore, activation was seen after treatment with thyrotropin and Gpp(NH)p even carried out in the absence of Mg2+ or at 4 °C. However, the full activation by Gpp(NH)p required Mg2+, hormone, and elevated temperature. These observations suggest that there appears to be two types of nucleotide interaction responsible for the Gpp(NH)p activation in human thyroid membrane; one type seen in the absence of hormone may represent the system uncoupled from hormone receptor, while the fully coupled hormone-sensitive adenylate cyclase accounts for the second type of interaction which requires the presence of hormone. 相似文献
Rat lenses in organ culture which are exposed to bovine rod outer segments (ROS) or to the major fatty acid of ROS, docosahexaenoic acid, are impaired in their ability to accumulate radiolabeled compounds which lenses normally accumulate by active processes. The extent of lens damage correlates well with the extent of lipid peroxidation in the culture medium as assessed by the thiobarbituric acid assay. Addition of vitamin E to the medium inhibits the effect on the lens while addition of Fe-ADP complexes potentiates the effect. Thus, the lens damage appears to be attributable to toxic species generated by peroxidation of the polyunsaturated lipid added to the culture medium. Toxic aldehyde products appear to be major mediators of the lens damage, since semi-carbazide, which avidly reacts with aldehydes, can protect lenses in this system. These findings may have relevance to the cataracts clinically associated with retinal degenerative diseases such as retinitis pigmentosa. The highly membranous photoreceptor cells are extremely rich in polyunsaturated lipid. Degeneration of these cells, which is the primary pathology in such diseases, would likely lead to peroxidation with generation of toxic products within the eye. Such products could potentially produce secondary damage to other ocular structures including the lens. 相似文献
Concanavalin A (Con A) or phytohemagglutinin activate a population of human circulating lymphocytes to exert suppressive functions. We found that supernates from the activated human lymphocytes suppress lymphocyte responses to Con A, the mixed lymphocyte reaction and pokeweed mitogen-induced IgM production. Mitogen stimulated suppressor lymphocytes, or their supernates, inhibit also the spontaneous proliferation of human retinoblastoma cells (Y-79 line) and primary cultures of human keratocytes. A correlation was always noted between the levels of inhibitory activities of the lymphocytes and their supernates. Furthermore, a good correlation was found between the levels of inhibition by the supernates of lymphocyte functions (proliferation and IgM production) and of the nonlymphoid cells' proliferation. Some of the properties of this suppressor factor(s) are: (i) produced only by the T-cell population; (ii) appears after 8 hr of Con A stimulation, peaks at 24 to 48 hr and declines later on; (iii) stable at 56 °C and labile to 70 °C; (iv) nondialyzable and present in the 40K–100K dalton fraction of a G-200 Sephadex column; (v) labile to pH 2 treatment. 相似文献
Peritoneal macrophages from Schistosoma mansoni-infected mice are activated both for nonspecific tumor cytotoxicity and for killing of skin-stage schistosomula in vitro. In the current study, mechanisms for induction of macrophage tumoricidal and schistosomulacidal activity have been compared. Examination of macrophages activated in vivo by BCG infection or C. parvum treatment, or in vitro by exposure to lymphokine prepared from antigen-stimulated BCG-immune spleen cells, showed that these effector functions were closely linked. Indeed, fractionation of lymphokine-rich supernatant fluids by Sephadex G-100 gel filtraction showed that activities responsible for induction of schistomula killing by inflammatory macrophages and for induction of tumoricidal activity cochromatographed as a single peak in the 50,000 MW region. Thus, development of macrophage-mediated cytotoxicity against these two extracellular (tumor cell or helminth) targets was coincident in several cell populations activated in vivo or in vitro. However, activation for tumoricidal and schistosomulacidal capacity appeared to be quantitatively dissociated in macrophages from mice with chronic schistosomiasis; those cells demonstrated low, yet significant, levels of larval killing ( to those of BCG or lymphokine-activated cells) but maximal levels of tumor cell cytotoxicity. Furthermore, cytotoxicity by peritoneal cells from S. mansoni-infected mice was not increased in vitro by exposure to lymphokine. Identification of this functional alteration in S. mansoni-activated cells may help to clarify the role of macrophages in the partial immunity against challenge infection which is demonstrated by mice with chronic primary S. mansoni infection. 相似文献
The extracellular chitinase produced by Serratia marcescens was obtained in highly purified form by adsorption-digestion on chitin. After gel electrophoresis in a nondenaturing system, the purified preparation exhibited two major protein bands that coincided with enzymatic activity. A study of the enzyme properties showed its suitability for the analysis of chitin. Thus, the chitinase exhibited excellent stability, a wide pH optimum, and linear kinetics over a much greater range than similar enzymes from other sources. The major product of chitin hydrolysis was chitobiose, which was slowly converted into free N-acetylglucosamine by traces of β-N-acetylglucosaminidase present in the purified preparation. The preparation was free from other polysaccharide hydrolases. Experiments with radiolabeled yeast cell walls showed that the chitinase was able to degrade wall chitin completely and specifically. 相似文献
A Beckman Airfuge has been employed for studying the interaction between lipids and the receptor for immunoglobulin E (IgE). For analytic experiments, samples were applied underneath a discontinuous sucrose gradient. After a 30-min centrifugation in a fixed-angle rotor, liposomes floated toward the top of the gradient whereas unincorporated receptor-IgE complexes remained at the bottom of the tube. Liposomes with incorporated receptors were also efficiently separated in the ACR-90 preparative rotor. These methods of "Airfuge flotation" can provide useful adjuncts to more traditional methods for density-gradient centrifugation especially when rapid analysis of small samples is desired. 相似文献
The l-thyroxine binding site in human serum thyroxine-binding globulin was investigated by affinity labeling with N-bromoacetyl-l-thyroxine (BrAcT4). Competitive binding studies showed that, in the presence of 100 molar excess of BrAcT4, binding of thyroxine to thyroxine-binding globulin was nearly totally abolished. The reaction of BrAcT4 to form covalent binding was inhibited in the presence of thyroxine and the affinity-labeled thyroxinebinding globulin lost its ability to bind thyroxine. These results indicate BrAcT4 and thyroxine competed for the same binding site. Affinity labeling with 2 mol of BrAcT4/mol of thyroxine-binding globulin resulted in the covalent attachment of 0.7 mol of ligand. By amino acid analysis and high voltage paper electrophoresis, methionine was identified as the major residue labeled (75%). Lysine, tyrosine, and histidine were also found to be labeled to the extent of 8, 8, and 5%, respectively. 相似文献
Murine peritoneal macrophages (M phi), activated in vivo or in vitro, remarkably inhibited the uptake of thymidine by a lens epithelial cell line, while resident M phi, or M phi induced by thioglycollate, exhibited much lower or no cytostatic capacity. The target cells were partially protected from the cytostatic activity by the anti-inflammatory agents indomethacin, aspirin, and dexamethasone, but not by lipoxygenase inhibitors. The protective activity of indomethacin and aspirin, but not of dexamethasone, was completely counteracted by prostaglandin E2 (PGE2). Yet, PGE2 alone has no effect on the uptake of [3H]thymidine by lens epithelial cells. PGE1 resembled PGE2 in its effect on this system, whereas PGA2, PGB2, or PGF2 alpha had no detectable activity. The counteracting effect of PGE2 was mimicked by dibutyryl cAMP or by cholera toxin, an agent which increases cAMP levels. These findings suggest that PGEs are not direct cytostatic agents, but rather, are essential mediators for the development of the cytostasis. Activated M phi did not lyse cells of the original lens epithelial cell line, but caused substantial cytolysis of cells of a subline derived from it. In contrast to its aforementioned effect on the cytostasis, PGE2 inhibited the cytolytic activity of M phi. Thus, this study provides a first demonstration in a single system of the opposite effects of PGEs on M phi activity on target cells, i.e., mediating the cytostasis and inhibiting the cytolysis. 相似文献
Under certain growth conditions, some strains of Escherichia coli accumulate toxic levels of methylglyoxal. This report characterizes a strain which synthesizes a mutant cAMP receptor protein in an adenylate cyclase deletion background. When cultured in glucose 6-phosphate minimal medium, this strain (222) was prematurely growth arrested due to methylglyoxal production; growth inhibition did not occur when the strain was grown in glucose minimal medium. A comparison of a variety of enzyme and cofactor levels in the related strains 222 (mutant) and 225 (wild-type) grown on either glucose or glucose 6-phosphate medium was carried out. The only difference found that might explain an increase in methylglyoxal accumulation was an elevated level of phosphofructokinase in strain 222 grown on glucose 6-phosphate. Since this enzyme activity probably limits hexose phosphate metabolism, it is suggested that growth inhibition in strain 222 may be due to increased production of triose phosphate, some of which is converted to methylglyoxal. 相似文献
A fluorometric procedure is described that can be used in the alkaline elution technique for the measurement of DNA damage in cells whose DNA is not, or cannot be, radioactively labeled. The procedure can be used for the measurement of DNA single-strand breaks, DNA-protein crosslinking, and DNA interstrand crosslinking, and possibly other DNA lesions produced in unlabeled cells. Although developed for the measurement of DNA damage in tissue-cultured cells, the technique is applicable to the measurement of DNA damage in cells isolated from tissues exposed to DNA damaging agents in vivo. 相似文献
Four hybridomas obtained from mice immunized with human adenocarcinomas of colon or stomach produce antibodies that bind specifically in solid-phase radioimmunoassay to the ceramide pentasaccharide that contains the lacto-N-fucopentaose III sequence of sugars. Binding of the antibodies to the glycolipid is inhibited by lacto-N-fucopentaose III, but not by structurally related oligosaccharides. The antibodies bind to glycolipids of erythrocytes, granulocytes, and certain normal and malignant tissues. 相似文献
The relationship between the appearance of cells producing antibody to tetanus toxoid (TT) in the circulation and the serum titers of anti-TT IgG following booster immunization has been studied. It was found that cells producing anti-TT antibody can be detected in the circulation in a hemolytic plaque assay using sheep red blood cells (SRBC) coated with TT by the chromic chloride method. In symmetric inhibition studies using cells from TT or keyhole limpet hemocyanin (KLH)-immune donors, the homologous antigen inhibited 100% of the PFC with no cross-inhibition. Thus, the plaque-forming cells (PFC) detected in this assay are specific for the immunizing antigen. No evidence of polyclonal B-cell activation in response to TT was found, as shown by a failure to detect any PFC against unmodified or KLH or human serum albumin-treated SRBC. In addition, the increase in total Ig-secreting cells observed in a staphylococcal protein A reverse hemolytic plaque assay was always accounted for by the number of anti-TT antibody-producing cells observed. The peak number of anti-TT antibody-producing cells varied between donors, but the kinetics of their appearance was highly reproducible--none before Day 5, peak numbers between Days 6 and 8, and a sharp decline with only rare anti-TT Ig-secreting cells in the circulation by Day 15 postimmunization. Anti-TT antibody-producing cells appeared in the circulation prior to any detectable increase in serum anti-TT antibody titers, and following the disappearance of PFC from the circulation, there was no further increase in serum IgG anti-TT levels. These observations demonstrate a marked specificity of B-cell activation on boosting with a recall antigen, and a parallelism between the appearance of activated B cells in the circulation and of IgG anti-TT synthesis by the subject as a whole. 相似文献
In vitro T-cell requirements for and modulation of human B-cell responses were studied in individuals immunized in vivo to the protein antigen keyhole limpet hemocyanin or tetanus toxoid. T cells were required for antibody synthesis in both antigen-driven and pokeweed mitogen (PWM)-driven cultures. T cells were separated into T4+ and T8+ subpopulations using monoclonal antibodies, and their modulation of antibody synthesis was studied. T4+ cells functioned as helper cells in both antigen-driven and PWM-driven cultures in a dose-dependent manner. Whereas T8+ cells suppress both total and specific immunoglobulin secretion in PWM-stimulated cultures, in antigen-stimulated cultures T8+ cells do not suppress unless activated by another cell population present in peripheral blood mononuclear cells (PBMNC). This cellular requirement was further investigated by prestimulation of cells prior to addition to optimally stimulated antigen-driven cultures of PBMNC or B cells, monocytes, and helper T cells. No suppression of these optimally stimulated cultures was seen when T8+ cells were precultured with antigen or PWM. However, after 3-5 days preculture of total T cells with PWM or antigen and then selection of T4+ cells, these cells were able to induce fresh autologous T8+ cells to suppress optimally stimulated antigen-driven cultures. Addition of a precultured mixture of T8+ cells with 20% T4+ cells also resulted in antigen-induced suppression. These data indicate that T8+ cells can suppress antigen-driven cultures but require the presence of preactivated T4+ cells for induction of this suppression of antigen-specific T-cell-dependent human B-cell responses. 相似文献
Incubation of turkey erythrocyte membranes with cholera toxin and [32P]NAD caused toxin-dependent incorporation of 32P into a 42,000 Mr peptide which could be distinguished from toxin-independent 32P incorporation into other membrane proteins. The radiolabeled 42,000 Mr peptide could be extracted from the membranes using Lubrol PX. When toxin-treated membranes were incubated with isoproterenol and GMP before detergent solubilization, the 42,000 Mr labeled peptide was adsorbed by GTP-γ-agarose which, with the same conditions, adsorbed the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide and guanine nucleotide regulatory protein activity were coeluted from the affinity matrix by guanylyl-β,γ-imidodiphosphate, GDP, and GMP. Guanosine 5′-O-(2-thiodiphosphate), an analog of GDP which blocks guanine nucleotide- and fluoride-stimulated adenylate cyclase activity, caused elution of labeled peptide which exhibited no regulatory protein activity. Our data support the view that the 42,000 Mr peptide is part of the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide allows identification of both active and inactive regulatory protein and should be useful in monitoring the purification of the regulatory protein from turkey erythrocytes. 相似文献
The development of antigen-specific T lymphocyte lines and clones has greatly facilitated the investigation of T-cell recognition of and response to foreign antigens. In the present study, human antigen-specific helper T cell lines and clones which are completely independent of exogenous interleukin-2 (IL-2) have been developed by cyclic restimulation with the soluble antigen keyhole limpet hemocyanin (KLH) to which the T cell donor had previously been immunized. These T cells uniformly bear the OKT4 phenotype and were shown to require both histocompatible antigen-presenting cells (APC) and antigen for optimal proliferation. The T cell line was composed of a highly antigen-specific and clonable T cell population. Following four cycles of antigen stimulation, limiting dilution cloning analysis showed a Poisson distribution of clonable T cells with a precursor frequency of 0.62, and from 88 to 92% of viable clones were specific for the stimulating antigen. Individual clones were obtained which recognized KLH with either DR 1 (one parental Ia haplotype of the donor) or DR 2 (the other parental Ia haplotype) allogeneic APC, but not both. Following stimulation with KLH, the T cell clones produced IL-2. Peak amounts of IL-2 were assayable in the first 6 to 24 hr after stimulation. In contrast, virtually no IL-2 was detectable in supernatants at 72 to 96 hr, suggesting autoutilization by the proliferating T cells. In addition, some clones were also capable of producing both B cell growth factor and IL-2 following KLH stimulation. These IL-2-independent T cells appeared to be derived from a discrete Leu 8-negative subclass of T4+ cells and expressed the full complement of Ia antigen of the donor. Thus, soluble antigen-specific human helper T cell clones have been produced which can be maintained in the absence of exogenous IL-2, elaborate their own growth factors and other immunoregulatory lymphokines, and show fine DR-related restriction to either one or the other parental DR haplotypes in antigen-stimulated proliferative responses. 相似文献
Mice were orally administered sheep erythrocytes (SRBC) in a regimen previously known to produce systemic tolerance to SRBC. Cellular interactions and movement from the gut-associated lymphoid tissue (GALT) to the spleen were found to occur using both in vivo and in vitro transfer systems. The cell in the GALT which initiates the suppression circuit migrates from the GALT to the spleen shortly after contacting antigen. This cell is a T suppressor-inducer (Tsi) cell which interacts with splenic lymphocytes to induce the formation of an effector T suppressor cell (Ts). The Tsi and Ts can be separated from each other by their differential sensitivities to cyclophosphamide. In addition, the Tsi can be separated from other GALT T cells by its inability to bind the lectin, peanut agglutinin. Thus, cell migration and cellular interaction among T cells must occur to result in orally induced tolerance. 相似文献
In this study, the question of whether human leukocyte-derived and fibroblast-derived interferon had an effect on prostaglandin metabolism in human peripheral blood mononuclear cells has been considered. Both leukocyte- and fibroblast-derived interferon were potent inhibitors of mononuclear cell prostaglandin synthesis at low physiological concentrations. Inhibition required a minimum incubation of 1 hr. Interferon had no effect on release of arachidonic acid; synthesis of hydroxy fatty acids was slightly increased. 相似文献
The hepatic pattern of synthesis of the apolipoprotein A-I isoforms has been analyzed in the rat. After isolated livers were perfused with defibrinated rat blood and [3H]leucine, the radioactivity associated with apolipoprotein A-I and other apolipoproteins was determined following two-dimensional gel electrophoresis of the perfusate d < 1.21 g/ml lipoprotein fraction. In rat serum, apolipoprotein A-I is a polymorphic system consisting of two major isoproteins and a series of minor species. Following liver perfusion, 72% of the radioactivity associated with apolipoprotein A-I isoproteins was recovered in the more acidic and quantitatively less abundant of the two major isoforms. Only 8% was associated with the major apolipoprotein A-I isoform, and similar or lower amounts were found in the other minor isoproteins. These results are consistent with the concept that, in the rat, the major apolipoprotein A-I isoforms differ in their pattern of biosynthesis and/or metabolism. 相似文献