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Analyzes the dynamic integration of green fluorescent protein (GFP)-coronin into the actin cytoskeleton, to examine the architectural dynamics of actin in live Dictyostelium amoebae. Experiments conducted; Observations noted; What the observations show.

Background: We have previously reported the design, pharmacological properties and imaging application of bradykinin (BK) B 2 receptor (B 2 R) ligands conjugated with fluorophores such as fluorescein derivatives at their N-terminus. To take advantage of the high penetration of infrared light into living tissues and their low autofluorescence in this region of the spectrum, additional probes conjugated with cyanine dye 7 (Cy7) were synthesized and characterized., Results: The antagonist B-9430 (D-Arg-[Hyp 3 ,Igl 5 ,D-Igl 7 ,Oic 8 ]-BK) and the agonist B-9972 (D-Arg-[Hyp 3 ,Igl 5 ,Oic 7 ,Igl 8 ]-BK) were N-terminally extended with the infrared fluorophore Cy7, producing the peptides B-10665 and B-10666, respectively. Pharmacological studies indicated that the agonist B-10666 lost much affinity for the B 2 R vs. the parent peptide, whereas the antagonist B-10665 better retained its potency vs. B-9430 (competition of [ 3 H]BK binding to human B 2 R, contractility of the human isolated umbilical vein for which potency losses were more important in each case). Both probes stained HEK 293 cells that expressed the B 2 R-green fluorescent protein (GFP) construction in a specific manner (confocal microscopy) and with very extensive co-localization of the green and infrared fluorescence in either case. The agonist B-10666 at 100 nM promoted the endocytosis of B 2 R-GFP in live cells, but not the antagonist version at 10-25 nM. The Cy7-labeled peptides did not label cells expressing the β 2 -adrenoceptor-GFP construction. B-10665 at low nanomolar concentrations was an effective probe for the recombinant B 2 Rs in cytofluorometry and macroscopic imaging of cell wells (IVIS imaging system operated for infrared fluorescence detection)., Conclusions: Despite a propensity for non-specific binding when used at high concentrations and limited sensitivity, Cy7-conjugated peptidase-resistant B 2 R ligands support original imaging and cytofluorometric applications.

Corrections to the articles published in previous issues are presented including "New Tools for Classification and Monitoring of Autoimmune Diseases," and "Screening: CyTOF--The Next Generation of Cell Detection."

Fluorescence imaging for detection of non-muscle-invasive bladder cancer is based on the selective production and accumulation of fluorescing porphyrins—mainly, protoporphyrin IX—in cancerous tissues after the instillation of Hexvix®. Although the sensitivity of this procedure is very good, its specificity is somewhat limited due to fluorescence false-positive sites. Consequently, magnification cystoscopy has been investigated in order to discriminate false from true fluorescence positive findings. Both white-light and fluorescence modes are possible with the magnification cystoscope, allowing observation of the bladder wall with magnification ranging between 30×for standard observation and 650×. The optical zooming setup allows adjusting the magnification continuously in situ. In the high-magnification (HM) regime, the smallest diameter of the field of view is 600 micronsand the resolution is 2.5 micronswhen in contact with the bladder wall. With this cystoscope, we characterized the superficial vascularization of the fluorescing sites in order to discriminate cancerous from noncancerous tissues. This procedure allowed us to establish a classification based on observed vascular patterns. Seventy-two patients subject to Hexvix®fluorescence cystoscopy were included in the study. Comparison of HM cystoscopy classification with histopathology results confirmed 3233(97) cancerous biopsies and rejected 1720(85) noncancerous lesions. [ABSTRACT FROM AUTHOR]

Abstract: Autoantibodies against red blood cell antigens are considered the diagnostic hallmark of AIHA: Direct antiglobulin test (DAT) completed by cytofluorometry and specific diagnostic monoclonal antibodies (mAbs) allow for a better understanding of autoimmune hemolytic anemia (AIHA) triggers. Once B-cell tolerance checkpoints are bypassed, the patient loses self-tolerance, if the AIHA is not also caused by an possible variety of secondary pathogenic events such as viral, neoplastic and underlying autoimmune entities, such as SLE or post-transplantation drawbacks; treatment of underlying diseases in secondary AIHA guides ways to curative AIHA treatment. The acute phase of AIHA, often lethal in former times, if readily diagnosed, must be treated using plasma exchange, extracorporeal immunoadsorption and/or RBC transfusion with donor RBCs devoid of the auto-antibody target antigen. Genotyping blood groups (www.bloodgen.com) and narrowing down the blood type subspecificities with diagnostic mAbs help to define the triggering autoantigen and to select well compatible donor RBC concentrates, which thus escape recognition by the autoantibodies. [Copyright &y& Elsevier]

The article presents a study on bimolecular fluorescence (BiFC) approach for the visualization of caspase 2 activation of single cells. In the process, caspase 2 dimerization induces activation from the fused carboxy-terminal and amino-terminal parts of venus in which caspase 2 brings the venus protein parts in contact resulting a flourescent signal that reflects real-time activation. Moreover, the authors validated the technique on studying the activation of initiator caspase in real time.

The chemokine (C-C motif) receptor CCR5 and its ligand CCL5 play key roles in the intra-articular recruitment of peripheral blood mononuclear cells (PBMC) in rheumatoid arthritis (RA). Therefore, using quantitative cytofluorometry, we followed T4 cell surface CCR5 density in 27 subjects with RA before and after treatment with the anti-CD20 monoclonal antibody rituximab. We observed low T4 cell surface CCR5 densities before treatment, which correlated positively with disease activity, as determined using a disease activity score evaluated on 28 joints (DAS 28), and negatively with CCL5 mRNA concentrations in PBMC, contrasting with a high proportion of intracellular CCR5 molecules, a pattern compatible with ligand-induced CCR5 internalization. At 3 months post-treatment, CCL5 mRNA expression in PBMC declined, whereas T4 cell surface CCR5 densities increased proportionally to the decrease in DAS 28. Thus, peripheral blood T4 cell surface CCR5 density is a good surrogate marker of RA activity and of the efficiency of anti-CD20 therapy. [ABSTRACT FROM AUTHOR]

Medullary carcinoma of the thyroid requires aggressive treatment because of its potential to metastasize and because of the current limitations of preoperative localization and systemic therapy. If these tumors could be made to fluoresce in vivo with tagged fluorophore antibodies against tumor antigens, surgeons would be able obtain additional information in the operating room to facilitate a more complete resection. Based on the success of our previous work in breast and colon cancer models, we conducted an animal study of in vivo tumor fluorescence of a human medullary thyroid cell line in which bright tumor fluorescence is visible during dissection. To accomplish this, we used an inexpensive and commercially available handheld, blue (470 nm), light-emitting diode flashlight and filtered goggles (520 nm). This procedure, which we call the fluorescent antibody-assisted surgical technique (FAAST), is easy to perform, requires no complex or expensive technical equipment, and has the potential to be applied to a wide variety of tumors. To the best of our knowledge, this is the first experiment of its kind to be reported in the literature. [ABSTRACT FROM AUTHOR]

Discusses cell selection strategies to improve the success of gene therapy for blood disorders. Information on the green fluorescent labeling technique; Obstacles to gene therapy.

Karyotype analysis by pulsed-field gel electrophoresis of several field isolates of Claviceps purpurea showed extensive variation in size and number of chromosomes. Analysis of F1-ascospore-lines of a cross between two field isolates showing chromosome-length polymorphisms (CLPs) indicate that recombination during meiosis contributes considerably to this variability. In addition, cytofluorometrical estimation of DNA content of single nuclei indicate that the C. purpurea strains vary in their ploidy level: most strains probably are ± diploid, some appear to be haploid, others ± triploid (with varying degrees of aneuploidy). Benomyl treatment (as used, e.g. for ‘haploidization’ in the parasexual cycle of imperfect fungi) of some field isolates confirmed that the differences in 4′-6-diamidino-2-phenylindol (DAPI)fluorescence correspond to different ploidy levels: derivatives with lower nuclear DNA content could be isolated from the putatively nonhaploid strains; a minimal value (even after repeated benomyl treatment) was obtained, corresponding to the lowest value of field isolates, probably representing the haploid status. Most of these strains are not impaired in fitness and aggressiveness and therefore can be used for the selection of recessive mutants and for functional molecular studies. [ABSTRACT FROM AUTHOR]

Focuses on the ability of the green fluorescent protein (GFP) to autocatalytically form a fluorescent pigment for light-harvesting proteins from natural amino acids present in the nascent protein. Preference of the approach over the attachment of beta-galactosidase to proteins and the provision of substrate for visualization; Spawning of fluorescence resonant energy transfer experiments.