Erdafitinib is an approved tyrosine kinase inhibitor that inhibits fibroblast growth factor receptor. It has been described as one of the potent anti-tumor drugs especially for the treatment of urothelial carcinoma. In this study, we have investigated the binding dynamics of erdafitinib with human serum albumin (HSA) using multiple spectroscopic techniques. The outcome of the results suggests the occurrence of static quenching during the interaction of HSA with erdafitinib which leads to the formation of non-fluorescent HSA-erdafitinib ground state complex. Formation of HSA-erdafitinib complex was also confirmed from the findings of absorption spectral analysis. The changes in microenvironment around hydrophobic domains (especially tryptophan and tyrosine) were deciphered from fluorescence spectroscopy which was further confirmed by synchronous spectral analysis. In order to gain insight into the binding site of erdafitinib in HSA, molecular docking combined with competitive displacement assay was performed. The modified form of Stern Volmer equation was used to estimate various binding parameters including number of binding sites. The findings are indicative of a single binding site (n = 1) with binding constant in the order of 104. The negative values of thermodynamic parameters like ΔG, ΔH and ΔS were suggestive of the binding reaction being spontaneous and exothermic, while the hydrogen bonds and Van der Waals interactions being the major forces present between HSA and erdafitinib. Circular dichroism spectral analysis revealed the alterations in the conformation of HSA structure and reduction in its α-helical content.Communicated by Ramaswamy H. Sarma
Aflatoxin B1 (AFB1), a potent mutagen, is synthesized by Aspergillus parasiticus and Aspergillus flavus. Human serum albumin (HSA) is a globular protein with diverse roles. As AFB1 is ingested with food and is transported in the body via blood, it becomes pertinent to comprehend the effect of the binding of this toxin on the structure and conformation of HSA, which may help to get insight into the toxic effect of the exposure of the mycotoxin. In this study, multi-spectroscopic approaches have been used to evaluate the binding efficiency of AFB1 with both the native HSA (nHSA) and the glycated HSA (gHSA). Steady-state fluorescence spectroscopy reveals the static type of fluorescence quenching in the fluorescence emission spectra of nHSA and gHSA in the presence of AFB1. The binding constant (Kb) is calculated to be 6.88 × 104 M-1 for nHSA, while a reduced Kb value of 2.95 × 104 M-1 has been obtained for gHSA. The circular dichroism study confirms the change in the secondary structure of nHSA and gHSA in the presence of AFB1, followed by alterations in the melting temperature (Tm) of nHSA and gHSA. In silico computational findings envisaged the amino acid residues and bonds involved in the binding of nHSA and gHSA with AFB1. The comprehensive study analyzes the binding effectiveness of AFB1 with nHSA and gHSA and shows reduced binding of AFB1 to gHSA.Communicated by Ramaswamy H. Sarma.As revealed by UV-absorption spectroscopy, the hyperchromic effect was more prominent in nHSA than gHSA in the presence of AFB1.The binding constant (Kb) obtained for the nHSA-AFB1 complex was 6.88 × 104 M−1, and the gHSA-AFB1 complex yielded Kb value of 2.95 × 104 M−1.Negative enthalpy change (ΔH) and entropy change (ΔS) suggested hydrogen bonding and van der Waals interaction as stabilizing forces of nHSA-AFB1 and gHSA-AFB1 complex.Site markers displacement assay suggested Sudlow’s site I as the binding site for AFB1 in nHSA and gHSA.Circular dichroism study showed that AFB1 induced secondary structural changes in nHSA and gHSA.Melting temperature (Tm) increased in nHSA and decreased in gHSA in the presence of AFB1.Molecular docking results confirmed Lys-195, Arg-222 and Arg-257 as hydrogen bonding residues in the nHSA-AFB1 complex and Arg-222 and Lys-199 residues were involved in hydrogen bonding in the gHSA-AFB1 complex.
The recent conference on Indoor Air Quality, Immunity and Health (June 1995) held at North Carolina State University, Raleigh, North Carolina, and cosponsored by the Cornell University Institute for Comparative and Environmental Toxicology, the Cornell Center for the Environment, and North Carolina State University provided an opportunity to examine a significant societal inhalation toxicology issue with appropriate scientific expertise from across the relevant disciplines. The program, which brought together leading national scientists as well as policy formulators, also included important policy perspectives for the discussion of scientific data application. This conference review by the organizing committee provides an overview of the proceedings and serves to introduce the following abstracts contributed by the presenters.
The effects of catecholamines (CA) on various chicken macrophage functions were examined. Macrophage monolayers were exposed to. 01,. 1,. 25, 1, 2, and 5 (μg/mL of dopamine (DA), norepinephrine (NE) and epinephrine (E) for 1 hr. All CA were toxic for macrophages at 1 -5 μg dose range resulting in 25-50% cell death. All CA at the. 1 and. 25 μg/mL level increased E. coli and sheep red blood cells (SRBC) phagocytosis by macrophages. the percentage of Fc-receptor positive macrophages increased after CA exposure. Prolonged exposure of macrophages (3 hr) reduced SRBC phagocytosis by DA-treated but not in NE-and E-treated macrophages. However, after 1 hr exposure and 3 hr recovery period, CA-induced changes were reversed in all but DA-treated cultures. Apomorphine and metoclopromide blocked DA whereas propranolol blocked NE and E effects suggesting specificity of the observed effects via catecholaminergic receptors on chicken macrophages. Dopamine and NE (.25 μg/mL) did not affect but E exposure enhanced LPS-induced tumoricidal factor production. These findings suggest that CA modulate chicken macrophage effector functions.
AbstractMacrophage cells isolated from the abdominal cavity of 21-day-old turkeys after a single injection of Sephadex suspension were used to quantitate the effects of direct in vitro exposure to deoxynivalenol (DON), 3-acetyldeoxynivalenol (3ac-DON), scirpentriol (STO), or 15-acetylscirpenol (15-MAS). Macrophage monolayers were established on glass surfaces and cells were exposed to graded levels of individual mycotoxins for 1 hour: DON, 20 -640 μ9/μ1 of culture; 3ac-DON, STO, 15-MAS, 20 -1280 μg/μ1 of culture. All four mycotoxins caused dose-related effects. A concentration of 50 μg/ml DON caused a significant decrease in macrophage adherence, phagocytosis of opsonized SRBC, and number of opsonized SRBC per macrophage; at 200 μg/ml, phagocytosis of unopsonized SRBC was decreased. There were also increasing percentages of damaged macrophages with increasing DON doses as indicated by morphological alterations. Linear decreases in macrophage viability on exposure to 3-acDON and STO were observed. Moreover, STO and 15-MAS decreased macrophage adherence to glass and 3-acDON, STO, and 15-MAS induced macrophage morphological alterations. This study suggests that trichothecene mycotoxins may be immunosuppressive by affecting viability, adherence and phagocytic potential of mononuclear phagocytic cells of young turkeys.
Abstract Rabbit antisera capable of detecting chicken fetal antigen (CFA) was prepared against 1‐day chick red blood cells (RBCs) and made specific by exhaustive adsorption with adult chicken peripheral RBCs (PRBCs) from the same strain. Microcytotoxicity was used to study the incidence of CFA on lymphocytes obtained from several organs at different developmental stages in the chicken. Lymphocyte‐associated CFA (LA‐CFA) determinants and erythrocytespecific CFA (ES‐CFA) determinants were distinguished through the use of adsorption analysis. The high incidence of CFA‐positive lymphocytes found in the fetal bursa and thymus was not equaled in the peripheral organs of the spleen, cecal tonsils, and gland of Harder. CFA expression on adult lymphocytes was restricted to the thymus and peripheral blood. It is suggested that these cells may represent a subpopulation of T lymphocytes. Adult spleen, cecal tonsils, and gland of Harder were virtually devoid of CFA‐bearing lymphocytes. At fetal developmental stages when greater than 94% of the bursal B cells were CFA‐positive, few, if any, of the highly differentiated Harderian B cells possessed CFA. It is suggested that LA‐CFA expression is dependent upon B cell differentiation and/or the bursa (central) vs gland of Harder (peripheral) microenvironment. The pattern of CFA expression on bursacytes is discussed in light of the properties of age resistance and bursal‐dependent target cells associated with virally induced lymphoid leukosis.
The objective of this study was to examine the hematopoietic cell proliferation and differentiation potential of growth factors produced by chicken macrophages. Bone marrow (BM) cells (25 × 103) from newly hatched B15B15K-strain Leghorn chicks were seeded in .5 mL serum-free semi-solid culture supplemented with 10% (vol/vol) of a conditioned medium (CM) from a chicken macrophage cell line, MQ-NCSU. The conditioned medium was obtained by culturing MQ-NCSU cells either in LM-HAHN (CMI) or RPMI-1640 (CMII) growth medium. The control cultures contained only LM-HAHN or RPMI medium. Bone marrow cells in the presence of CMI differentiated predominately into granulocyte colonies (Experiment 1 = 84±9.2; Experiment 2 = 105 ± 5). No colonies were observed in, the control cultures. Stimulation of MQ-NCSU cells with lipopolysaccharide (LPS) produced a CM that differentiated BM cells predominantly into macrophage colonies (122±16.3 in CMI and 92±5.6 in CMII). These data suggest that MQ-NCSU cells spontaneously secrete a factor with the potential to promote granulocyte differentiation. However, upon stimulation with LPS, the factor secreted had macrophage colony stimulation potential (M-CSF), which was similar in activity when compared with the activity of recombinant chicken myelomonocytic growth factor (r-cMGF). Another CM from chicken fibroblasts (FCM) was tested on BM cells from K-strain Leghorns and Arbor Acres × Arbor Acres broiler chicks. Data from three experiments showed that 25 × 103 BM cells from K-strain chicken yielded more macrophage and granulocytes colonies (82 ± 14) than those from broilers (56 ± 12). This study suggests that avian cytokines exhibit progenitor cell differentiation potential and that this activity is dependent upon the source of cytokines and their targets.
Synthesis of heat-shock proteins (HSP) in chicken macrophages, in response to thermal and nonthermal stressors, was determined. Cornell K-strain 6-wk-old White Leghorn females were injected with Sephadex and approximately 42 h later subjected to elevated temperatures in order to achieve a core body temperature (CBT) of 44 C. Peritoneal macrophages were isolated at 30 and 60 min after heat treatment. A parallel group of chickens, maintained at the normal CBT of 41C, was used as controls and peritoneal macrophages were isolated after 60 min of treatment. For in vitro study of HSP response, cells of a chicken macrophage cell line (MQ-NCSU) were subjected to 45 C ambient temperature to produce heat shock (HS, thermal stress), lipopolysaccharide (LPS, 15 μg), and lead acetate (nonthermal stress) exposure for varying time periods. The HSP profiles of macrophages following various treatments were determined by one- and two-dimensional gel electrophoresis. The results showed that macrophages isolated from the 44 C CBT group synthesized HSP90, HSP70, HSP23, and a heat-inducible P32 protein. This HSP synthesis profile was similar to the HSP expression by MQ-NCSU cells exposed in vitro to 45 C conditions. Exposure to MQ-NCSU cells to lead acetate induced the same four proteins previously expressed by macrophages after in vivo or in vitro heat treatment. Two-dimensional analysis of lysates from cells treated with LPS, HS, or LPS plus HS treatments revealed a doublet protein molecule (70a and 70b) with identical molecular mass of 70 kDa. However, the pI value (isoelectric point) of 70b was higher (5.1) than that of 70a, which, along with HSP90 and HSP23, focused more toward the acidic side with a pI value of less than 4.6. The present study is the first to report pI profiles of chicken macrophage HSP. The in vitro and in vivo studies suggest that chicken macrophages respond to thermal and nonthermal stressors by producing similar kinds of “stress proteins”.
Abstract Chick embryos were mutagenized in ovo in order to study developmentally related alterations in immune functions in survivors of this prenatal toxicant insult. In this experimental system, a single exposure of 6‐day chick embryos to 0.1 μg aflatoxin‐B 1 (AF‐B 1 ) in 10 μ1 of acetone was employed, and the control embryos received 10 μ1 of solvent alone. This dosage of AF‐B 1 administered to 6‐day embryos was found to increase the incidence of sister chromatid exchanges in blood cells approximately fivefold above the baseline observed in solvent controls. A second sham control, where no solvent was administered, was included in some experiments. The cell cycle times in blood increased slightly during the initial exposure to AF‐B 1 . However, a majority of the AF‐B 1 and acetone exposed embryos survived and hatched without incident. Losses occurred mainly in the latter part of embryogenesis. After hatching, no significant differences were observed in body weight between different treatment groups up to 26 weeks of age and no change in primary humoral immunity was detected. In contrast, two parameters of cell‐mediated immunity, graft vs host (GvH), and cutaneous basophil hypersensitivity (CBH) reactions were both depressed as a result of exposure to AF‐B 1 . The AF‐B 1 treatment group was significantly reduced in the GvH reaction compared with sham‐treated controls. In the CBH assay, AF‐B 1 ‐exposed chicks showed reduced immunity compared with acetone controls. These results suggest that long‐term selective immune depression can occur following embryonic exposure to AF‐B 1 .
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