White Blood Cell Rheology
Gordon D. O. Lowe in Clinical Blood Rheology, 2019
Experiments have also been performed with the use of cytochalasin B, which disrupts the actin microfilaments. In this case all three elements decrease. These results suggest that both the microfilaments and microtubules are important in governing the passive viscoelastic properties of the WBC; their disruption leads to a decrease in the viscoelastic coefficients of the cell. Furthermore, the results suggest that the microfilaments and the microtubules have different roles in the control of the viscoelastic properties of the cell. The microtubules apparently are not important in determining K1, whereas the disruption of microfilaments has an influence on all three elements. The interrelationship between the chemical and the physical properties of the cell needs to be further studied.
Continuous Monitoring of Superoxide Production by Phagocytes
Robert A. Greenwald in CRC Handbook of Methods for Oxygen Radical Research, 2018
Table 1 is a summary of results obtained in our laboratory using the continuous assay with various perturbations. Reversible activation of the superoxide generating system could be achieved with several different stimuli by the subsequent addition of a specific reagent: with Con A followed by α-methyl mannoside, with n-formyl-norleucyl-leucyl-phenylalanine (FNLP) followed by t-BOC FMLP and with arachidonic acid followed by albumin. EGTA prevented activation of the oxidase for all stimuli tested (with the exception of PMA) but only for arachadonic acid did it affect ongoing O2− production. Cytochalasin b had a number of effects on the assay. It had no effect on digitonin-, PMA- or arachidonic acid-stimulated superoxide production; however, it dramatically increased the rate of Con A-stimulated superoxide production. It appeared to increase the rate of STZ-stimulated superoxide production. This apparent increase, however, was secondary to more efficient detection of the superoxide produced. Finally, in the case of FMLP, cytochalasin b had no effect on the initial rate of O2− production, but dramatically prolonged the duration from 1 to 2 min to 5 to 10 min.
The αv Integrins
Yoshikazu Takada in Integrins: The Biological Problems, 2017
Integrin αvβ3 binds to vitronectin and fibronectin in a two-step process which is initially RGD-dependent and dissociable, followed by a non-RGD-dependent stabilization between the receptor and ligand.61 Non-RGD-containing sequences are required for molecular stabilization, since αvβ3 binding to a vitronectin-derived RGD-containing peptide (YPQVTRGDVFTMPED) failed to result in stabilization.61 Mild glutaraldehyde fixation of purified αvβ3 also resulted in failure of molecular stabilization after binding to vitronectin,61 suggesting receptor conformation is important in stabilization. Integrin αvβ3 has a 50-fold higher association rate constant for vitronectin as compared to that for the vitronectin-peptide, 9.0 × 103 s−1 M−1 vs. 1.7 × 102 s−1 M−1, respectively.61 Receptor-ligand stabilization occurred after pretreatment of M21 melanoma cells with cytochalasin B (an inhibitor of actin polymerization), suggesting molecular stabilization is independent of the actin-cytoskeleton.61 Taken together, these data suggest the mechanism of receptor-ligand stabilization involves conformationally dependent protein-protein interactions of the receptor and ligand. This concept is consistent with integrin receptor conformational changes postligand binding reported by other investigators.25,62
Cytotoxicity and genotoxicity of clothianidin in human lymphocytes with or without metabolic activation system
Published in Drug and Chemical Toxicology, 2019
Zülal Atlı Şekeroğlu, Vedat Şekeroğlu, Ebru Uçgun, Seval Kontaş Yedier, Birsen Aydın
Clothianidin (CHN) ((E)-1-(2-chloro-5-thiazolylmethyl)-3-methyl-2-nitroguanidine) was obtained from Sigma-Aldrich (Steinheim, Germany) (analytical standard >99%, CAS Number 210880-92-5). The chemical structure of CHN is shown in Figure 1. The test substance was dissolved in dimethyl sulfoxide (DMSO) supplied by Merck (Darmstadt, Germany, CAS 67-68-5). In the cultures without metabolic activation, Mitomycin-C (MMC) (Serva, Heidelberg, Germany, CAS 50-07-7) was used as positive control at 0.16 µg/ml for treatments. For cultures with metabolic activation, cyclophosphamide monohydrate (CP, Acros Organics, NJ, USA, CAS 6055-19-2) was used as a positive control at 45 µg/ml. They were dissolved in sterile distilled water. MMC is a clastogen without metabolic activation while CP is a clastogen requiring metabolic activation OECD (2014). Therefore, we used MMC for cultures without S9 mix and CP for cultures with S9 mix. Cytochalasin-B (Cyt-B) was obtained from Sigma (St. Louis, MO, CAS 14930-96-2) and colcemid was supplied by Biological Industries (Beit Haemek, Israel, Cat. No. 12-004-1). Human liver S9 pooled donors (20 mg/ml, product number 452961), NADPH Regenerating System Solution A (product number 451220) and NADPH Regenerating System Solution B (product number 51200) were supplied by Corning® Gentest™ (Tewksbury, MA, USA). Other chemicals used for fixation and staining were obtained from Merck (Darmstadt, Germany). All test solutions were freshly prepared prior to each experiment.
The platelet shape change: biophysical basis and physiological consequences
Published in Platelets, 2019
Alexander E. Moskalensky, Alena L. Litvinenko
The right-hand side of the equilibrium condition (threshold force qcr) can be changed by alteration of the marginal band and/or the actin network. We should note that Eq. (1) does not account for the actin network, which stabilizes the discoid shape. The influence of activation signaling on the actin network is mediated by gelsolin, a molecule that severs actin filaments [32]. Although the actin network is not the major factor maintaining the discoid platelet shape, it does contribute to disk-to-sphere transformation. Delayed response of platelets to stimuli was reported for patients with hereditary gelsolin-related amyloidosis (AGel amyloidosis), a systemic disorder related with G654A or G654T mutation in the gene coding for gelsolin [33]. Altered platelet rounding was also shown in transgenic gelsolin-null mice [34]. In these experiments, later stages of shape change were inhibited with the use of cytochalasin B. This agent interferes with actin polymerization, and was used to study the importance of this process for platelet function [35,36]. Preincubation with cytochalasin prevent pseudopodia formation and also the recruitment of integrin αIIbβ3 to platelet surface [37], which together result in altered aggregation, although P-selectin expression is increased [38]. This data suggests that the actin network additionally elevates the threshold value qcr in the resting platelet, but its disruption upon activation permits the shape change to occur.
Chronic low dose exposure of hospital workers to ionizing radiation leads to increased micronuclei frequency and reduced antioxidants in their peripheral blood lymphocytes
Published in International Journal of Radiation Biology, 2019
Zothan Siama, Mary Zosang-zuali, Annie Vanlalruati, Ganesh Chandra Jagetia, Kham Suan Pau, Nachimuthu Senthil Kumar
The blood samples were collected by venipuncture from each volunteer of both groups in individual sterile heparinized tubes. The lymphocyte culture was carried out according to the method described earlier (Jagetia et al. 2001). Briefly, the blood was allowed to sediment and the buffy coat was collected in individual sterile glass tubes. Usually, 106 nucleated cells were inoculated into sterile glass tubes containing RPMI-1640 medium, 10% fetal calf serum and phytohemagglutinin as the mitogen. The cells were allowed to grow for the next 44 h in a humidified atmosphere of 5% CO2 in air at 37 °C. Cytochalasin B was added at a final concentration of 5 μg/ml to block cytokinesis and cells were allowed to grow for another 28 h (Fenech and Morley 1985). Cells were harvested at the end of 72 h after initiation of the lymphocyte culture by centrifugation. A mild hypotonic solution was added to the cell pellet so as to retain the cell membrane. Cells were then fixed in freshly prepared Carnoy’s fixative (methanol: acetic acid, 3:1). The cell suspension was placed onto pre-cleaned coded blinded slides to avoid observer`s bias and spread by air blowing. The cells were stained with acridine orange and scored under a fluorescence microscope (DM 2500, Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany) by two individuals. Usually, a total of 1000 binucleate cells (BNC) with well-preserved cytoplasm were scored from each individual for the presence of micronuclei (MN) according to the criteria described earlier (Fenech et al. 2003).