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Results for “Functional characterization”

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At least 73 records · Page 4Linked to original sources

PubMed1979

Functional characterization of drug-induced experimental papillary necrosis.

The functional expression of papillary necrosis was investigated with a model of drug-induced papillary necrosis. Bromoethylamine hydrobromide (BEA) administration to rats uniformly resulted in the development of papillary necrosis. All studies were performed 24 hours after BEA administration with the exception of the electrolyte balance studies, which were performed during the 72 hours after the induction of papillary necrosis. GFR was not different between BEA-treated and sham rats. BEA-treated rats had a significantly lower maximal urine osmolality and free water reabsorption than did sham rats. Renal tissue concentrations of sodium, potassium, and water were not different between BEA-treated and sham rats. During water diuresis, free water clearance was not significantly different between the two groups. During sodium bicarbonate administration, maximal bicarbonate reabsorption and urine-blood Pco2 gradient (at comparable urine bicarbonate concentrations) were not significantly different between the two groups. During sodium sulfate infusion, there was no difference in minimum urine pH, ammonium excretion, and net acid excretion between chronically acidotic BEA-injected and sham rats. In rats on "zero" sodium intake, BEA administration resulted in a significant increase in urine flow and sodium excretion, whereas sham rats remained in sodium balance. In rats with restriction of both sodium and chloride, BEA administration resulted in a significant wastage of sodium, chloride, and calcium. There was no difference in potassium excretion between BEA-treated and sham rats during hydropenia, bicarbonate administration, sodium sulfate infusion, or ingestion of a normal potassium diet. When potassium intake was restricted to "zero," BEA-treated rats developed potassium wastage; when potassium intake was increased to 21 mEq/day, BEA-treated rats had a significantly lower potassium excretion than did sham rats. These findings may result from alterations in collecting duct transport, but damage to deep medullary structures may also contribute.

Animals↗

PubMed1979

Functional characterization of mouse T lymphocytes with IgM-Fc receptors. I. Studies on ADCC and helper cell function.

The function of mouse T lymphocytes with receptors for IgM-Fc was analyzed by using immunofluorescence and a column separation method. The cellular uptake of FITC-IgM was inhibited with unlabeled Fc5 mu. IgM-FcR+ cells were active in ADCC against IgM-sensitized target cells, but not against IgG-coated target cells. The cells mediating ADCC with IgM antibody were shown to be distinct and physically separable from those mediating ADCC with IgG antibody. Helper T lymphocytes for humoral antibody formation in a hapten-carrier system were shown to be IgM-FcR+ and Fc-IgG-.

Animals↗

PubMed1985

Antigenic profile and functional characterization of human peritoneal macrophages.

The antigenic profile and the functional properties of human peritoneal macrophages have been analyzed by using a panel of monoclonal antibodies (MoAb) and functional assays. All peritoneal macrophages were stained by the anti-class I HLA MoAb Q6/64. Between 40 and 100% of the cells were stained by the anti-HLA-DR + DP MoAb Q2/80, Q5/6, and Q5/13; approximately 80% of the cells were stained by the anti-HLA-DQ MoAb BT3.4, and about 95% were stained by the anti-macrophage MoAb OKM1. Peritoneal macrophages were not stained by the anti-dendritic cells MoAb Ki-M4 or by MoAb to T cell subsets, although all of the MoAb were reactive with the appropriate substrates. More than 60% of the cells expressed Fc receptors and C3 receptors, and displayed phagocytic activity. Peritoneal macrophages were effective in stimulating autologous and allogeneic lymphocytes and in presenting soluble antigens to T cells. These reactions were blocked by the anti-HLA-DR + DP MoAb Q5/13, but were not affected by the anti-dendritic cells MoAb Ki-M4 or by the anti-class I HLA MoAb Q6/64. These results suggest that human peritoneal macrophage preparations, without detectable contamination with dendritic cells, can induce proliferation of autologous and allogeneic T cells, and that class II HLA antigens play a significant role in these phenomena.

Antigen-Presenting Cells↗

PubMed1991

Structure, expression and in vitro functional characterization of a novel RNA binding zinc finger protein from Xenopus.

Large multigene families of zinc finger proteins are expressed in vertebrates. One way of approaching their function is to characterize their structure, expression and biochemical properties. XFG 5-1 is a Xenopus zinc finger protein which is widely transcribed in oocytes, embryos and adult tissues. It carries a novel, non-finger repeat structure, which is common to a subfamily of Xenopus zinc finger proteins. The bacterially expressed protein exhibits specific RNA homopolymer binding activities with the zinc finger domain being sufficient for this ability. These findings suggest that XFG 5-1 serves a general biological function involving its RNA binding capacity.

Amino Acid Sequence↗

PubMed1984

Functional characterization of human alloreactive T cell clones.

We have investigated the functional activity of alloreactive T cell clones, generated by limiting dilutions of MLC blasts. Clones displaying helper, suppressor, or cytotoxic functions (against Class I or Class II HLA antigens) were identified, using proliferative, 51Cr-release, and hemolytique-plaque induction assays. To determine whether functionally distinct clones share determinants that trigger the AMLC reaction we have tested the capacity of selected clones to cross-stimulate each other in secondary AMLC. Our data suggest that AMLC stimulatory determinants are related to common structure antigens of the T cell receptor for alloantigen, since helper clones, for example, (TLC #19) can induce memory responses to suppressor (TLC #209) as well as cytotoxic (TLC #207) clones and vice versa. The AMLC system seems well suited for studying network regulations within the T cell compartment.

B-Lymphocytes↗

PubMed1979

Functional characterization of left ventricular segmental responses during the initial 24 h and 1 wk after experimental canine myocardial infarction.

Characterization of the temporal evolution of resting segmental function and inotropic reserve after coronary occlusion may be important in evaluating attempts to salvage ischemic but non-necrotic myocardium. Accordingly, we chronically implanted up to six pairs of pulse-transit piezoelectric crystals in the left ventricular myocardium of dogs to measure segmental wall thickness. Segments were separated into groups according to the loss of net systolic thickening (NET) at 5 min postocclusion of the left anterior descending coronary artery in awake, unsedated dogs. Group 1 included segments with NET values of 67--100+ (percent control); group 2 between 67 and 0; and group 3 less than 0 (paradoxical motion). 5 min after coronary occlusion, group 1 NET was 92 +/- 5% (SEM) although significant decreases occurred in NET in group 2 (36 +/- 4%) and group 3 segments (-33 +/- 5%). Between 5 min and 24 h after coronary occlusion, no further significant changes occurred in NET in groups 1, 2, and 3 crystals. Some segments underwent further functional deterioration between 24 h and 1 wk after left anterior descending coronary artery occlusion, although no overall change occurred in segments with mild to moderate ischemic dysfunction. Segments with NET less than 0 at 24 h, on the other hand, exhibited a reduction in aneurysmal bulging between 24 h and 1 wk from -41 +/- 10 to -23 +/- 11% (n = 12, P = 0.02). Inotropic reserve was assessed with postextrasystolic potentiation (PESP) in 14 dogs, and with infusions of dopamine (11 dogs), and isoproterenol (13 dogs). PESP was the most potent intervention and produced a significant augmentation in NET in group 2 crystals at 1, 2, 4, 6,8, and 24 h after coronary occlusion but only at 1 and 2 h in NET in group 3 crystals. Thus, following experimental coronary occlusion, the evolution of ischemic segmental dysfunction is dynamic and variable. A significant degree of inotropic reserve, as assessed by PESP, dopamine, and isoproterenol, exists in segments with moderate ischemic dysfunction for 24 h but for only 2 h after coronary occlusion in those segments with the most severe ischemic dysfunction. In addition, at least some segmental sites with mild to moderate ischemic dysfunction at 24 h deteriorate further between 24 h and 1 wk after experimental coronary occlusion.

Animals↗

PubMed1983

Phenotypic and functional characterization of murine B lymphocyte precursors isolated from fetal and adult tissues.

Murine fetal liver and adult bone marrow cells identified by monoclonal 14.8 antibody were enriched on antibody-coated polystyrene petri dishes. Cell surface immunoglobulin (sig)-bearing cells were depleted before this enrichment procedure, and the resulting preparations of 14.8+, slg- cells were characterized as to morphology, immunoglobulin gene expression, and functional potential in vivo and in vitro. All cells with detectable mu chains of IgM in the cytoplasm (cmu) were found to be included in the 14.8+ population. The enriched cells did not contain significant numbers of committed granulocyte-macrophage progenitor cells or putative hemopoietic stem cells. Selected cells from 16-day fetal liver were large, a majority of the cells had a lobulated rather than a spherical nuclear outline, and less than 1% had detectable cmu. Enriched cells from 19-day fetal liver were on the average smaller than those from 16-day-gestation liver and had a more typical lymphoid morphology; 30% were cmu+. Adult bone marrow 14.8+, slg- cells were similar to 19-day fetal liver cells in morphology, and approximately half were cmu+. These selected precursor cells retained the capacity to mature in vivo and in vitro. Fetal and adult 14.8+, slg- cells were efficient in generating newly formed B cells in vivo, and this maturation step appeared to be dependent on the presence of microenvironmental accessory cells. However, the ability of positively selected cells to mature in vitro was markedly decreased, and this potential was not rescued by providing known sources of accessory cells. Possible reasons for this difference are considered. This technique for positively selecting cells has allowed us to directly compare for the first time B cell precursors from fetal and adult tissues and will be invaluable for resolution of the cell compartments in the differentiation of B lymphocyte precursors, in the study of accessory cells known to facilitate this process, in the definition of humoral factors which may act on pre-B cells, in the study of immunoglobulin gene rearrangements which take place during normal differentiation, and for further comparative studies of fetal and adult lymphopoiesis.

Animals↗

PubMed1989

Functional characterization of ecto-5'-nucleotidase-positive and -negative human T lymphocytes.

Functional studies were performed on human peripheral blood T lymphocytes stained with goat anti-5'-nucleotidase antibodies and separated into ecto-5'-nucleotidase (ecto-5'-NT)-positive and -negative populations using the FACSTAR fluorescence-activated cell sorter. On the average, ecto-5'-NT+ T cells contained 34 +/- 13% CD4+ and 55 +/- 15% CD8+ cells, whereas ecto-5'-NT-T cells contained 65 +/- 12% CD4+ and 23 +/- 8% CD8+ cells. Staining with anti-5'-NT antibodies did not significantly alter the ability of unseparated T cells to proliferate in response to PHA or PMA, or in a MLR. However, prior incubation with anti-5'-NT antibodies did inhibit the ability of irradiated T cells to provide help for PWM-stimulated Ig synthesis by as much as 55%. In five separate experiments, ecto-5'-NT-T cells demonstrated an equal or better ability to incorporate [3H]TdR after PHA stimulation or in a MLR, as compared with ecto-5'-NT+ T cells. Similarly, ecto-5'-NT- T cells were not diminished in their ability to provide help for autologous B cells in a PWM-driven system. Clearly, the inability of ecto-5'-NT- T cells from patients with a variety of immunodeficiency diseases to function in these assays cannot be explained solely by their lack of ecto-5'-NT activity. In contrast, ecto-5'-NT-positive and -negative T cells showed markedly different dose-response curves for proliferation in response to PMA. Ecto-5'-NT+ T cells responded to lower doses of PMA (1.0 ng/ml) than did ecto-5'-NT- T cells and showed a two- to eight-fold greater rate of [3H]TdR incorporation at 3 to 10 ng of PMA per ml. Ecto-5'-NT+ T cells may have a protein kinase C that is more accessible or more easily activated or may utilize an alternate pathway of activation when stimulated with low concentrations of PMA.

5'-Nucleotidase↗

PubMed1986

Phenotypic and functional characterization of the circulating NK compartment in hairy cell leukaemia.

A phenotypic and functional analysis of the circulating natural killer (NK) cell population was carried out in a series of patients with hairy cell leukaemia (HCL). The overall mean NK activity of both the mononuclear and T cell fractions was reduced compared to that of normal controls (466 lytic units (lu) v 573 lu and 226 lu v 381 lu, respectively), though this difference did not reach statistical significance (P less than 0.05). Individual analysis of the data showed that in five out of 15 and in seven out of 16 cases the K562 killing by the mononuclear and T cells respectively was below the lowest s.d. limit for normal subjects. This reduced NK function was associated with a decreased ability of the effector cells to bind the target. The NK response to exogenous human leucocyte interferon was also generally depressed in cases with a low basal NK activity. The functional studies were complemented with the evaluation of the membrane expression of NK associated antigens. The percentage of circulating T cells recognized by the monoclonal antibody (MoAb) Leu-7 was significantly higher (P less than 0.001) in HCL than in normal blood (25.2% +/- 10.2 v 11.9 +/- 5.9 s.d.). However, the reactivity with two other NK-related MoAb, Leu-11 and AB8.28, was significantly lower (7.1% +/- 6.9 and 9.8% +/- 8.5; P less than 0.002) than with Leu-7 and moderately reduced compared with that of normal circulating T cells (11.7% + 6.1 & 12% + 5.5). These findings suggest that in a proportion of patients with HCL there is an impairment of the NK compartment, which may contribute towards the occurrence of the infective complications which are the primary cause of death in this disease.

Antibodies, Monoclonal↗

PubMed1978

Separation and functional characterization of human neutrophil subpopulations.

Human neutrophils have been considered to be a functionally homogeneous population of cells. We have developed a density sedimentation technique for separation of neutrophils into two populations based on their ability to form rosettes with IgG-coated human erythrocytes (7SEA). Under the experimental conditions 80% +/- 4.3% of normal human peripheral blood neutrophilis form rosettes. Functionally rosette-forming neutrophils are more adherent to nylon wool, able to phagocytize more 14C-labeled Staphylococcus aureus, more efficient in killing S. aureus, and more responsive to endotoxin-activated human serum in a 51-cr chemotaxis assay that the non-rosette forming neutrophils. However, there is no difference among neutrophil subpopulations' ability to phagocytize latex particles. Paired samples of exudate neutrophils from cutaneous abscess fluid and peripheral neutrophils from three patients were investigated for their ability to form 7SEA rosettes. In each case exudate neutrophils contained greater than 96% rosette-forming neutrophils, whereas peripheral blood contained the normal 80% ( less than 0.01). Thus we show that peripheral blood contains at least two distinct populations of neutrophils. However, an essentially homogeneous neutrophil population is present in cutaneous exudate fluid.

Animals↗

PubMed1990

Functional characterizations of the androgen receptor confirm that the molecular basis of androgen action is transcriptional regulation.

In an effort to understand the molecular basis of androgen action in the prostate, we isolated androgen receptor (AR) cDNA from rat ventral prostate cells and analyzed the transcriptional regulatory activity of the encoded protein in a cotransfection assay. We found that AR is capable of inducing chloramphenicol acetyltransferase activity more than 20-fold using the mouse mammary tumor virus LTR as a source of androgen response elements. This induction was observed in both monkey CV1 cells and human HeLa cells, neither of which contains endogenous functional AR, and was entirely dependent on added androgens. Deletion mapping studies showed that carboxy-terminal deletions of approximately 250 amino acids convert AR into a constitutive activator of transcription. In addition, a chimeric receptor protein containing the amino-terminus and DNA-binding domains of AR fused to the previously defined ligand domain of the glucocorticoid receptor was found to be fully functional based on dexamethasone-induced chloramphenicol acetyltransferase activity. Our results support the prediction that androgens modulate rates of transcriptional initiation, suggesting that posttranscriptional effects of androgens are secondary responses. Moreover, these data reveal that, like other steroid receptors, AR contains a number of distinct regulatory regions important for normal activity. The isolation and characterization of fully functional AR sequences will facilitate the use of molecular genetics to study complex androgen responses in target tissues such as the prostate.

Androgens↗

PubMed1985

[3H]Adenosine binding to rat mast cells--pharmacologic and functional characterization.

Rat serosal mast cell adenosine receptors were characterized by [3H]adenosine binding to cell membrane particulates, and functional changes in mast cell mediator release and cyclic AMP levels were assessed, utilizing various adenosine analogs. [3H]adenosine binding to sonicated mast cell membrane preparations at 0 degrees C in the presence of deoxycoformycin is linear with initial cell number, rapid and reversible. The cells display 16,400 +/- 1600 high affinity [3H]adenosine binding sites/cell, equivalent to 118 fmol bound/mg protein, with an equilibrium dissociation constant of 27.97 +/- 3.0 nM. Competition studies reveal that adenosine greater than 2-chloroadenosine greater than NECA greater than L-PIA greater than D-PIA in competing for [3H]adenosine binding sites and that aminophylline and cromolyn sodium also bind to the putative receptor. Adenosine and its analogs, NECA, and L-PIA, appear to activate adenylate cyclase in resting mast cells by raising cyclic AMP, suggesting an Ra cell surface adenosine receptor subtype; these same analogs potentiate mast cell B-hexosaminidase release stimulated by specific antigen. The identification of rat mast cell [3H]adenosine binding sites whose stimulation augments resting cell cyclic AMP levels and antigen-induced mediator release suggests that these receptors may be important in the biochemical mechanisms of allergic diseases. The ability to assess the number and affinity of mast cell adenosine receptors will enable one to monitor receptor alterations during pharmacologic manipulation and in disease states.

Adenosine↗

PubMed1990

Biochemical and functional characterization of 8-10C5 (gp120,90), a novel molecule on the surface of bovine T leucocytes.

An unclustered molecule, 8-10C5, present on bovine leucocytes, was characterized using a monoclonal antibody (mAb) prepared at the University of Wisconsin-Madison, and the functional and biochemical characteristics of that molecule compared to defined molecules. This molecule was present on a large percentage of peripheral blood cells (84%), including T and B lymphocytes, monocytes and neutrophils, but was absent from immature thymocytes. mAb 8-10C5 blocked proliferation to alloantigen and bovine herpesvirus-1; however, antibody to the 8-10C5 molecule was unable to stimulate proliferation alone or in combination with PMA or a cross-linking agent. These results indicate that this molecule has a role in T-cell activation, but probably not signal transduction. mAb 8-10C5 was unable to block cytotoxic T lymphocyte (CTL) or natural killer (NK)-like lysis. mAb 8-10C5 identifies a non-covalently bound heterodimer present on most peripheral lymphocytes with a molecular weight (MW) of 120,000 for the alpha chain (10% glycosylation) and 90,000 MW for the beta chain (16% glycosylation). The beta chain is an acidic molecule (pI = 4.5-5.0). Biochemical studies included the CD2 and CD5 molecules and further demonstrated their similarity to equivalent molecules in other species, confirming the phylogenetic importance of these molecules in immune responses. This functional and biochemical information will allow further characterization of these molecules at the RNA and DNA levels.

Animals↗

PubMed1989

Structural and functional characterization of human immunodeficiency virus tat protein.

Site-directed mutagenesis was used to identify functional domains present within the human immunodeficiency virus (HIV) tat protein. Transient cotransfection experiments showed that derivatives of tat protein with amino acid substitutions either at the amino-terminal end or at cysteine residue 22, 37, 27, or 25 were no longer able to transactivate HIV long terminal repeat-directed gene expression. Incubation of Tat expressed in Escherichia coli with zinc demonstrated that both authentic Tat and cysteine mutation derivatives could form metal-protein complexes. The tat proteins that contained alterations within the cluster of positively charged amino acid residues retained their ability to transactivate gene expression, albeit at markedly reduced levels. Indirect immunofluorescence showed that the authentic tat protein and the amino-terminal and cysteine substitution mutants all localized in the nucleus, with accumulation being most evident in the nucleolus. In contrast, nuclear accumulation was greatly reduced with the basic-substitution mutations. Consistent with this result, a fusion protein that contained amino acids GRKKR, derived from the basic region, fused to the amino-terminal end of beta-galactosidase also accumulated within the nucleus. These results demonstrate that the 14-kilodalton tat protein contains at least three distinct functional domains affecting localization and transactivation.

Amino Acid Sequence↗

PubMed1984

[Chemical and functional characterization of lymphokines].

Macrophages are heterogeneous with respect to a number of constitutive and inducible functions. In order to study the underlying biological principle, a bone marrow liquid culture system was adopted in which bone marrow cells proliferate and differentiate into macrophages. It was found that maturing macrophages express various constitutive or inducible functions in an ordered sequence. The kinetics of their appearance and disappearance are dependent on the proliferative activity of macrophages. Macrophages in "late" G-1 of the cell cycle express constitutive functions like plasminogen activator production and are inducible by bacterial lipopolysaccharides, Poly I:C and lymphokines to release interferons. The response to lymphokines like migration inhibitory factor (MIF) and chemotactic factors is also transiently expressed during maturation. Using purified MIF, its influence on proliferation, differentiation and activation of macrophages was investigated. The changes induced were monitored following the expression of marker enzymes and of phenotype associated cell surface antigens using monoclonal antibodies. The results showed that functional changes induced by MIF on macrophages are limited and are not related to certain macrophage activating activities (MAF). As determined by flow cytofluorometry, transglutaminase expression and proliferation is consistently down-regulated by MIFs. This together with the shift and the expression of surface antigens indicates that MIFs provide a differentiation signal for a "young" macrophage to become more mature.

Animals↗

PubMed1989

Development and functional characterization of T cell lines from canine Peyer's patches.

We have propagated concanavalin A-stimulated cells from canine Peyer's patches in vitro in the presence of interleukin-2 (IL-2). The cells were characterized as T cells by determination of their phenotype and by functional assays. They are IL-2 dependent and respond to IL-2 of murine, primate and canine origin. The long-term cultured cells provided help for immunoglobulin production by purified autologous B cells and suppressed IgG production by nonseparated autologous peripheral blood mononuclear cells.

Animals↗

PubMed1991

Functional characterization of an abnormal factor XII molecule (F XII Bern).

An 18-year-old healthy woman was found to have cross-reacting material (CRM)-positive factor XII (F XII) deficiency, F XII clotting activity was less than 0.01 U/mL, whereas F XII antigen was 0.11 U/mL. An F XII inhibitor was excluded. To partially characterize the molecular defect of the abnormal F XII, immunologic and functional studies were performed on the proposita's plasma. The abnormal F XII was a single chain molecule with the same molecular weight (80 Kd) and the same isoelectric points (pl, 5.9 to 6.8) as normal F XII. Dextran sulfate activation of the proposita's plasma showed no proteolytic cleavage of F XII even after 120 minutes, whereas F XII in pooled normal plasma, diluted 1:10 with CRM-negative F XII-deficient plasma, was completely cleaved after 40 minutes. Adsorption to kaolin was identical for both abnormal and normal F XII. In the presence of dextran sulfate and exogenous plasma kallikrein, the abnormal F XII was cleaved with the same rate as normal F XII. However, kallikrein-cleaved abnormal F XII was not able to cleave factor XI and plasma prekallikrein, in contrast to activated normal F XII. Thus, these studies show that the functional defect of this abnormal F XII, denoted as F XII Bern, is due to the lack of protease activity of the kallikrein-cleaved molecule. Therefore, the structural defect is likely to be located in the light chain region of F XII, containing the enzymatic active site.

Adsorption↗

PubMed1989

Morphologic and functional characterization of human peripheral blood T cells expressing the T cell receptor gamma/delta.

The morphologic and functional characteristics of cells freshly isolated from human peripheral blood and bearing a T cell receptor (TcR) gamma/delta were analyzed. Cell preparations highly enriched for TcR gamma/delta+ cells were obtained by treatment of E rosette-forming lymphocytes with anti-CD4 and anti-CD8 monoclonal antibodies (mAb) and complement. These preparations consisted of 64-82% TcR gamma/delta+ lymphocytes, as indicated by the sum of cells reacting with the BB3 and A13 mAb which define two distinct, nonoverlapping, TcR gamma/delta+ cell subsets in the peripheral blood. TcR gamma/delta cells were able to form conjugates with the natural killer-sensitive K-562 and with the natural killer-resistant HL-60-R tumor cell lines. The cytochemical localization of lysosomal acid hydrolases showed that 95%-98% of the cells in the TcR gamma/delta+ preparations had the morphologic features of granular lymphocytes. Moreover, electron microscopy analyses showed that TcR gamma/delta+ cells had electron-dense granules dispersed in the cytoplasm and a variety of smooth vesicles, a morphology identical to that of other CD3- or CD3+ granular lymphocyte subsets. Freshly isolated TcR gamma/delta+ cells were unable to lyse K-562 and natural killer-resistant targets, such as HL-60-R and P815. However, low levels of target cell lysis were observed upon triggering of the effectors by anti-CD3 TcR mAb or by lectin. After short-term culture with interleukin 2, TcR gamma/delta+ cells acquired a strong cytolytic activity against K-562 and HL-60-R target cells in the absence of triggering stimuli, and also displayed high levels of cytolytic activity against P815 in the presence of anti-CD3/TcR mAb.

Antigens, Differentiation, T-Lymphocyte↗