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Although the glycoprotein (GP) Ib-IX-V complex andFc␥RIIA are distinct platelet membrane receptors, previous studies have suggested that these structures may be co-localized. To determine more directly the proximity of GP Ib-IX-V and Fc␥RIIA, we assessed the effects of anti-GP Ib␣ monoclonal antibodies on Fc␥RIIA-mediated platelet aggregation and on the direct binding of polymeric IgG to human platelets. In addition, we directly examined the proximity of Fc␥RII and GP Ib-IX-V using flow cytometric fluorescence energy transfer and immunoprecipitation studies. Preincubation of platelets with either of two monoclonal antibodies (AN51 or SZ2) directed against GP Ib␣ completely blocked platelet aggregation by polymeric IgG. Similarly, these antibodies totally inhibited platelet aggregation by two strains of viridans group streptococci known to induce aggregation via Fc␥RIIA. In addition, AN51 and SZ2 significantly reduced the binding of polymeric IgG to washed fixed platelets. When assessed by flow cytometry, significant levels of bidirectional energy transfer were detected between Fc␥RIIA and GP Ib␣, indicating a physical proximity of less than 10 nm between these receptors. This energy transfer was not due to high receptor density, because no homoassociative energy transfer was seen. Moreover, immunoprecipitation of Fc␥RIIA from platelet lysates also co-precipitated GP Ib␣. These results indicate that GP Ib␣ and Fc␥RIIA are co-localized on the platelet membrane and that this association is not random. The glycoprotein (GP)1 Ib-IX-V complex and Fc␥RIIA are distinct platelet membrane receptors for von Willebrand factor and polymeric IgG, respectively. The GP Ib-IX-V complex contains four polypeptides (GP Ib␣, GP Ib␤, GP IX, and GP V), which are present in a stoichiometry of 2:2:2:1 on the platelet plasma membrane (1). The precise configuration by which these peptides form a functional receptor is unknown, although more than one copy of each polypeptide per receptor complex is probable (1). This receptor mediates platelet adhesion to sites of blood vessel injury by binding von Willebrand factor in the subendothelium and participates in platelet activation by thrombin by providing a high affinity binding site for this agonist. In contrast to the GP Ib-IX-V complex, the platelet receptor for the Fc portion of IgG, Fc␥RIIA, is much simpler in structure, consisting of a single transmembrane polypeptide. This receptor is responsible for the aggregation and activation of platelets induced by immune complexes, opsonized bacteria, and certain antibodies that bind other platelet surface proteins (2).Although GP Ib-IX-V and Fc␥RIIA are structurally unrelated, a functional interaction between these receptors has been suspected for some time. Moore et al. reported that both polymeric IgG and IgG Fc fragments could inhibit platelet aggregation induced by von Willebrand factor and ristocetin (3). The reciprocal phenomenon was also observed, i.e. prior incubation of platelets with von Willebrand factor and ristocetin inhibited plate...

Alterations of neutrophil functions by tobacco products may play a central role in the pathogenesis of periodontal diseases and several smoking-related systemic diseases. In the present study, we examined the in vitro effects of cigarette smoke on neutrophils at times and concentrations that may be encountered during smoke exposure. We measured the level of smoke exposure in the in vitro system by measuring the levels of nicotine and comparing these to levels in the oral cavity in smokers before and after smoking. We examined both the unstimulated and stimulated release of 2 oxidative burst products: superoxide (O-2) and hydrogen peroxide (H2O2). Salivary washings were collected from 7 smokers (> 1 pack/day) before smoking a cigarette. Immediately after they smoked a cigarette, a second set of washings was collected. In vitro exposure to smoke involved incubating aliquots of neutrophils in phosphate-buffered saline for 1 to 5 minutes. Nicotine and cotinine levels were quantitated using gas chromatography, with detection by electron impact mass spectrometry. Peripheral neutrophils were isolated from medically healthy non-smoking volunteers via a double-density gradient technique and incubated in vitro with whole cigarette smoke for 0 to 5 minutes. Phorbol myristate acetate (PMA; 10(-7) M) was used to stimulate half of the cell aliquots. Superoxide generation was assessed through the superoxide dismutase (SOD) inhibitable reduction of ferricytochrome c. H2O2 production was assessed through the H2O2-dependent breakdown of dichlorofluorescin diacetate to its fluorophore and measured by flow cytometry. There was a marked elevation in salivary nicotine concentration from before smoking (mean: 80.8 ng) to after smoking (mean 1,685 ng/mL). In the in vitro smoke box system, there was a time-related elevation in nicotine from 1 to 5 minutes (50-->136 ng/mL). In PMA-stimulated cells exposed to smoke, there was a time-related inhibition of both superoxide and H2O2 production. However, in unstimulated cells exposed to smoke, there was a time-related increase in the release of superoxide and H2O2. A novel finding in unstimulated cells exposed to smoke was that there appeared to be 2 distinct populations of cells--one of "high" H2O2 producers and one of "low" H2O2 producers. The proportion of high H2O2 producers increased relative to smoke exposure. The relative production of H2O2 in the unstimulated high producers was comparable to PMA-stimulated cells at 5 minutes. This release of superoxide and H2O2 in unstimulated cells exposed to smoke may alter the pathogenic processes both in periodontal diseases and other systemic diseases.

Troglitazone is a potent agonist for the peroxisome proliferator-activated receptor-gamma (PPARgamma) that is a ligand-activated transcription factor regulating cell differentiation and growth. PPARgamma may play a role in thyroid carcinogenesis since PAX8-PPARgamma1 chromosomal translocations are commonly found in follicular thyroid cancers. We investigated the antiproliferative and redifferentiation effects of troglitazone in 6 human thyroid cancer cell lines: TPC-1 (papillary), FTC-133, FTC-236, FTC-238 (follicular), XTC-1 (Hürthle cell), and ARO82-1 (anaplastic) cell lines. PPARgamma was expressed variably in these cell lines. FTC-236 and FTC-238 had a rearranged chromosome at 3p25, possibly implicating the involvement of the PPARgamma encoding gene whereas the other cell lines did not. Troglitazone significantly inhibited cell growth by cell cycle arrest and apoptotic cell death. PPARgamma overexpression did not appear to be a prerequisite for a response to treatment with troglitazone. Troglitazone also downregulated surface expression of CD97, a novel dedifferentiation marker, in FTC-133 cells and upregulated sodium iodide symporter (NIS) mRNA in TPC-1 and FTC-133 cells. Our investigations document that human thyroid cancer cell lines commonly express PPARgamma, but chromosomal translocations involving PPARgamma are uncommon. Troglitazone, a PPARgamma agonist, induced antiproliferation and redifferentiation in thyroid cancer cell lines. PPARgamma agonists may therefore be effective therapeutic agents for the treatment of patients with thyroid cancer that fails to respond to traditional treatments.

Background: The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry. Methods: We generated chimeras in which cyan fluorescent protein (CFP) was separated by amino acid linkers of different sizes from yellow fluorescent protein (YFP) and used them to calibrate the cell-by-cell flow cytometric FRET measurements carried out on two different duallaser flow cytometers. Then, CFP-Kip1 was coexpressed in yeast cells with YFP and cyclin-dependent kinase-2 (Cdk2) and served as a positive control for FRET measurements, and CFP-Kip1 coexpressed with a random peptide fused to YFP was the negative control. Results: We measured donor, direct, and sensitized acceptor fluorescence intensities and developed a novel

Insufficient or dysregulated energy metabolism may underlie diverse inherited and degenerative diseases, cancer, and even aging itself. ATP is the central energy carrier in cells, but critical pathways for regulating ATP levels are not systematically understood. We combined a pooled clustered regularly interspaced short palindromic repeats interference (CRISPRi) library enriched for mitochondrial genes, a fluorescent biosensor, and fluorescence-activated cell sorting (FACS) in a high-throughput genetic screen to assay ATP concentrations in live human cells. We identified genes not known to be involved in energy metabolism. Most mitochondrial ribosomal proteins are essential in maintaining ATP levels under respiratory conditions, and impaired respiration predicts poor growth. We also identified genes for which coenzyme Q10 (CoQ10) supplementation rescued ATP deficits caused by knockdown. These included CoQ10 biosynthetic genes associated with human disease and a subset of genes not linked to CoQ10 biosynthesis, indicating that increasing CoQ10 can preserve ATP in specific genetic contexts. This screening paradigm reveals mechanisms of metabolic control and genetic defects responsive to energy-based therapies.

To study the role of the glycoprotein (GP) Ibalpha cytoplasmic domain in the mobility of the GP Ib-IX complex within the plasma membrane and in its ability to bind vWf, we established eight cell lines expressing GP Ib-IX complexes (these complexes lack GP V but function normally as receptors for vWf) that contain either wild-type GP Ibalpha or one of a series of GP Ibalpha truncation mutants missing different lengths of the cytoplasmic domain. To test the mobility of these complexes within the plasma membrane, we used the technique of fluorescence recovery after photobleaching after labeling them with a fluorescein-conjugated anti-GP Ibalpha monoclonal antibody. Fluorescence recovery within a bleached area on the cell surface was evaluated by scanning the cell surface with a low-intensity laser for 3 min after bleaching and then extrapolating the recovery values to infinite time. Fluorescence recovery in cells expressing wild-type GP Ibalpha was negligible. However, when only six amino acids were removed from the GP Ibalpha carboxyl terminus (t604 mutant, polypeptide length of 604 vs 610 residues for wild-type GP Ibalpha), complex mobility increased greatly, as judged by a more rapid recovery of fluorescence in the bleached area (48% recovery). The mobility increased further in the t594 mutant and remained approximately the same through the t534 mutant (55-67% recovery). A further increase in mobility was observed with the t518 mutant (>80% recovery), which lacks almost all of the GP Ibalpha cytoplasmic domain. The ristocetin-dependent binding of the mutant cell lines was also evaluated. Binding of vWf to cells expressing any of the mutant complexes was markedly lower than that to cells expressing the wild-type complex. These studies demonstrate that the cytoplasmic domain of GP Ibalpha fixes the position of the GP Ib-IX complex on the platelet surface and that this orientation is an important determinant of the complex's ability to bind vWf.

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