Pulse fluorimetric study of labelled actin—DNase I complex

Ikkai, Takamitsu; Mihashi, Koshin; Kouyama, Tsutomu

doi:10.1016/0014-5793(80)81090-8

Cited by 9 publications

(2 citation statements)

References 11 publications

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“…The time constants of the anisotropy decay for the slow component were approximately 20 ns and changed with OEG linker length only slightly ( θ 2 in Table 2 ), and the anisotropy decay amplitude ( r 1 and r 2 ) noticeably changed with OEG linker length. The time constants of anisotropy decay that were previously reported were somewhat longer than our result of 20 ns: 30 ns for G-actin labeled with dansyl or 5- ({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid (IAEDANS) at 25°C [ 30 ], 45.5 ns for G-actin labeled with IAEDANS at 25°C [ 31 ], and 27.53 ns and 30.43 ns for Ca- and Mg-G-actin, respectively, with IAEDANS at 22.7°C [ 32 ]. The difference of these time constants from the present value may have occurred, probably because these studies used the zero-time anisotropy during the analyses in different manners as the present study and the use of a low zero-time anisotropy value in the least squares fitting often tends to yield a longer time constant.…”

Section: Resultscontrasting

confidence: 54%

Rotational motion of rhodamine 6G tethered to actin through oligo(ethylene glycol) linkers studied by frequency-domain fluorescence anisotropy

2015

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Investigation of the rotational motion of a fluorescent probe tethered to a protein helps to elucidate the local properties of the solvent and protein near the conjugation site of the probe. In this study, we have developed an instrument for frequency-domain fluorescence (FDF) anisotropy measurements, and studied how the local properties around a protein, actin, can be elucidated from the rotational motion of a dye tethered to actin. Rhodamine 6G (R6G) was attached to Cys-374 using newly-synthesized R6G-maleimide with three different oligo(ethylene glycol) (OEG) linker lengths. The time-resolved anisotropy decay of R6G tethered to G-actin was revealed to be a combination of the two modes of the wobbling motion of R6G and the tumbling motion of G-actin. The rotational diffusion coefficient (RDC) of R6G wobbling was ~0.1 ns−1 at 20°C and increased with OEG linker length. The use of the three R6G-actin conjugates of different linker lengths was useful to not only figure out the linker length dependence of the rotational motion of R6G but also validate the analyses. In the presence of a cosolvent of glycerol, although the tumbling motion of G-actin was retarded in response to the bulk viscosity, the wobbling motion of R6G tethered to actin exhibited an increase of RDC as glycerol concentration increased. This finding suggests an intricate relationship between the fluid properties of the bulk solvent and the local environment around actin.

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Section: Resultscontrasting

confidence: 54%

Rotational motion of rhodamine 6G tethered to actin through oligo(ethylene glycol) linkers studied by frequency-domain fluorescence anisotropy

2015

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“…The slides were triple-labeled for DNA by using 15 ,uM Hoechst 33258 (20), for p185 by using the TAl antibody (21) directly conjugated to Texas red, and for p300 by using the M344 mouse monoclonal antibody (17) and a three-stage sequence using biotin-conjugated goat antibody to mouse IgGl as secondary antibody and fluorescein-labeled avidin (22). With 14 of the tumors and 5 of the controls a second triple-labeled slide was prepared, labeling for DNA as above, for EGFR by using AB-1 mouse monoclonal antibody (Oncogene Science) and the same visualization system as was used with M344, and for G-actin by using DNase I (Molecular Probes) directly conjugated to Texas red (23). A corresponding negative control slide omitting primary antibodies was also prepared.…”

Section: Methodsmentioning

confidence: 99%

Alterations in phenotypic biochemical markers in bladder epithelium during tumorigenesis.

Rao

Hemstreet

Hurst

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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Phenotypic biochemical markers of oncogenesis and differentiation were mapped in bladder biopsies to investigate changes that occur in bladder tumorigenesis and to identify markers for increased bladder cancer risk. Touch preparations from biopsy specimens from 30 patients were obtained from tumors, the adjacent bladder epithelium, and random distant bladder epithelium. Markers, including DNA ploidy, epidermal growth factor receptor (EGFR), and oncoproteins, were quantified in individual cells by using quantitative fluorescence image analysis. Cluster analysis revealed the markers fell into three independent groups: (0) G-actin and EGFR; (it) ploidy, cytology, and p185 (HER-2/neu oncoprotein) (ERBB2); and (iil) p300, a low-grade tumor antigen. Each marker displayed a gradient of abnormality from distant field to adjacent field to tumor. Different patterns for each marker suggested a developmental sequence of bladder cancer oncogenesis; G-actin was altered in 58% of distant biopsies (vs. 0/6 normals, P < 0.001), ploidy and cytology were altered in <20% of distant fields and =80% of tumors, and the other markers were intermediate. Patterns of EGFR and p185 suggest lowand high-grade tracks diverge early (P < 0.05 by MannWhitney U test for EGFR and ANOVA for p185). In conclusion, this study shows that a sequence of phenotypic changes accompanies development and progression of bladder cancers. Biochemical alterations in cells of the bladder field are often detectable before abnormal pathology, and markers previously thought to be limited to tumors were found in the field. The hierarchy of expression may be useful in identifying high-risk patients, assessing completeness of response to therapy, and monitoring and predicting recurrence.Decreasing future deaths from bladder cancer will require a strategy of prevention, based in part upon identification of individuals at risk for invasive metastatic disease well before it becomes symptomatic. Recognition that clinical cancer is the end-point of the underlying disease of carcinogenesis (1) suggests that appropriate intervention with individuals determined to be at risk could prevent development of lifethreatening disease.The concept of "field cancerization" or "field disease," introduced in 1953 (2), suggests that the whole field of tissue is exposed to carcinogen and is at increased risk for developing cancers, even though individual lesions are of clonal origin. The field disease theory as applied to bladder cancer has provided a central organizing theory for understanding these multifocal, frequently recurrent tumors (3-5). The normal bladder, or any other solid organ, represents a complex ecosystem ofinteracting epithelial and stromal cells, and years are required for the progressive subversion of growth and differentiation controls (6-8) to result in eventual emergence of cells capable of at least partial autonomousThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" i...

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Intermediate endpoint biomarkers for chemoprevention

et al. 1992

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The understanding of intermediate endpoint biomarker expression in relation to the sequential events in bladder tumorigenesis establishes a useful approach for evaluating chemopreventive agents. Biomarkers may be genotypic or phenotypic and function as biomarkers of susceptibility, exposure, effect, or disease. This paper reviews several years of research on biomarkers and their use in monitoring chemoprevention therapy. In initial animal experiments, mice were dosed with Kbutyl-N(4-hydroxybutyl)nitrosamine (OH-BBN) while co-administering K(4-hydroxypheny1)retinamide (4-HPR).4-HPR did not statistically reduce tumor incidence, but did affect tumor differentiation and, consequently, nuclear size and DNA ploidy. These results suggest that nuclear size and ploidy may function as intermediate endpoint biomarkers of effect for oncogenesis and that epigenetic as well as genetic mechanisms may be primary in the oncogenic process. Early biomarkers of effect which occur prior to genetic effects or chromosome aberration may portend a higher probability of being modulated by differentiating agents such as retinoids. In v i m studies demonstrated that RPMI-7666 cells cultured with a phorbol estertumor promoter (1 2-Otetradecanoyl-phorbol-13-acetate) could be redifferentiated with 13-cis-retinoic acid and dimethyl sulfoxide (DMSO). F-actin, a cytoskeletal biomarker with a presumed function in the epigenetic mechanisms of carcinogenesis, could also be normalized in HL-60 cells treated with 4-HPR or DMSO.A clinical evaluation of F-actin in patients with varying degrees of risk confirmed the value of F-actin as a differentiating biomarker useful for bladder cancer risk assessment. The clarification of when the phenotypic changes of F-actin occur in the oncogenic process was achieved when a variety of biochemical changes were mapped in the patients with bladder cancer. These studies confirmed that G-actin, a reciprocal form of F-actin, is increased relatively early in bladder cancer oncogenesis when multiple biomarkers are quantitated in the field, adjacent area, and the tumor. Comparison of each individual biomarker's expression from field, adjacent to tumor, and tumor, and subsequent cluster analysis of these biomarkers, indicated that the possible sequence of phenotypic expression of biomarkers in bladder cancer oncogenesis is from G-actin, to p300 antigen, to epidermal growth factor receptor (EGFR), to p185 (neu oncogene product), to DNA aneuploidy and, finally, to visual morphology. To date, a battery of three biomarkers, G-actin, M344, and DNA, with routine cytology has been used to monitor eleven patients receiving Bacillus Calmette-Guerin (BCG) immunotherapy and eight patients clinically free of bladder cancer (negative cytology and biopsy) who were treated with the differentiation agent, DMSO. These results indicate that G-actin may be a useful biomarker for evaluating the efficacy of chemopreventive agents.

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Pulse fluorimetric study of labelled actin—DNase I complex

Cited by 9 publications

References 11 publications

Rotational motion of rhodamine 6G tethered to actin through oligo(ethylene glycol) linkers studied by frequency-domain fluorescence anisotropy

Rotational motion of rhodamine 6G tethered to actin through oligo(ethylene glycol) linkers studied by frequency-domain fluorescence anisotropy

Alterations in phenotypic biochemical markers in bladder epithelium during tumorigenesis.

Intermediate endpoint biomarkers for chemoprevention

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