Добірка наукової літератури з теми "Single-cell membrane depolarization assay"

Glucose-induced insulin secretion by pancreatic β-cells depends on membrane depolarization and [Ca2+]i increase. We correlated voltage- and current-clamp recordings, [Ca2+]i measurements, and insulin reverse hemolytic plaque assay to analyze the activity of a thapsigargin-sensitive cationic channel that can be important for membrane depolarization in single rat pancreatic β-cells. We demonstrate the presence of a thapsigargin-sensitive cationic current, which is mainly carried by Na+. Moreover, in basal glucose concentration (5.6 mM), thapsigargin depolarizes the plasma membrane, producing electrical activity and increasing [Ca2+]i. The latter is prevented by nifedipine, indicating that Ca2+ enters the cell through L-type Ca2+ channels, which are activated by membrane depolarization. Thapsigargin also increased insulin secretion by increasing the percentage of cells secreting insulin and amplifying hormone secretion by individual β-cells. Nifedipine blocked the increase completely in 5.6 mM glucose and partially in 15.6 mM glucose. We conclude that thapsigargin potentiates a cationic current that depolarizes the cell membrane. This, in turn, increases Ca2+ entry through L-type Ca2+ channels promoting insulin secretion.

ABSTRACT Telavancin is an investigational lipoglycopeptide antibiotic currently being developed for the treatment of serious infections caused by gram-positive bacteria. The bactericidal action of telavancin results from a mechanism that combines the inhibition of cell wall synthesis and the disruption of membrane barrier function. The purpose of the present study was to further elucidate the mechanism by which telavancin interacts with the bacterial membrane. A flow cytometry assay with the diethyloxacarbocyanine dye DiOC2(3) was used to probe the membrane potential of actively growing Staphylococcus aureus cultures. Telavancin caused pronounced membrane depolarization that was both time and concentration dependent. Membrane depolarization was demonstrated against a reference S. aureus strain as well as phenotypically diverse isolates expressing clinically important methicillin-resistant (MRSA), vancomycin-intermediate (VISA), and heterogeneous VISA (hVISA) phenotypes. The cell wall precursor lipid II was shown to play an essential role in telavancin-induced depolarization. This was demonstrated both in competition binding experiments with exogenous d-Ala-d-Ala-containing ligand and in experiments with cells expressing altered levels of lipid II. Finally, monitoring of the optical density of S. aureus cultures exposed to telavancin showed that cell lysis does not occur during the time course in which membrane depolarization and bactericidal activity are observed. Taken together, these data indicate that telavancin's membrane mechanism requires interaction with lipid II, a high-affinity target that mediates binding to the bacterial membrane. The targeted interaction with lipid II and the consequent disruption of both peptidoglycan synthesis and membrane barrier function provide a mechanistic basis for the improved antibacterial properties of telavancin relative to those of vancomycin.

Cardiomyocytes derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) are increasingly used for in vitro assays and represent an interesting opportunity to increase the data throughput for drug development. In this work, we describe a 96-well recording of synchronous electrical activities from spontaneously beating hiPSC-derived cardiomyocyte monolayers. The signal was obtained with a fast-imaging plate reader using a submillisecond-responding membrane potential recording assay, FluoVolt, based on a newly derived voltage-sensitive fluorescent dye. In our conditions, the toxicity of the dye was moderate and compatible with episodic recordings for >3 h. We show that the waveforms recorded from a whole well or from a single cell-sized zone are equivalent and make available critical functional parameters that are usually accessible only with gold standard techniques like intracellular microelectrode recording. This approach allows accurate identification of the electrophysiological effects of reference drugs on the different phases of the cardiac action potential as follows: fast depolarization (lidocaine), early repolarization (nifedipine, Bay K8644, and veratridine), late repolarization (dofetilide), and diastolic slow depolarization (ivabradine). Furthermore, the data generated with the FluoVolt dye can be pertinently complemented with a calcium-sensitive dye for deeper characterization of the pharmacological responses. In a semiautomated plate reader, the two probes used simultaneously in 96-well plates provide an easy and powerful multiparametric assay to rapidly and precisely evaluate the cardiotropic profile of compounds for drug discovery or cardiac safety.

The melastatin (M) transient receptor potential (TRP) channel TRPM4 mediates pressure and protein kinase C (PKC)-induced smooth muscle cell depolarization and vasoconstriction of cerebral arteries. We hypothesized that PKC causes vasoconstriction by stimulating translocation of TRPM4 to the plasma membrane. Live-cell confocal imaging and fluorescence recovery after photobleaching (FRAP) analysis was performed using a green fluorescent protein (GFP)-tagged TRPM4 (TRPM4-GFP) construct expressed in A7r5 cells. The surface channel was mobile, demonstrating a FRAP time constant of 168 ± 19 s. In addition, mobile intracellular trafficking vesicles were readily detected. Using a cell surface biotinylation assay, we showed that PKC activation with phorbol 12-myristate 13-acetate (PMA) increased (∼3-fold) cell surface levels of TRPM4-GFP protein in <10 min. Similarly, total internal reflection fluorescence microscopy demonstrated that stimulation of PKC activity increased (∼3-fold) the surface fluorescence of TRPM4-GFP in A7r5 cells and primary cerebral artery smooth muscle cells. PMA also caused an elevation of cell surface TRPM4 protein levels in intact arteries. PMA-induced translocation of TRPM4 to the plasma membrane was independent of PKCα and PKCβ activity but was inhibited by blockade of PKCδ with rottlerin. Pressure-myograph studies of intact, small interfering RNA (siRNA)-treated cerebral arteries demonstrate that PKC-induced constriction of cerebral arteries requires expression of both TRPM4 and PKCδ. In addition, pressure-induced arterial myocyte depolarization and vasoconstriction was attenuated in arteries treated with siRNA against PKCδ. We conclude that PKCδ activity causes smooth muscle depolarization and vasoconstriction by increasing the number of TRPM4 channels in the sarcolemma.

3035 Background: CD37 is a tetraspan transmembrane family protein selectively expressed on normal and transformed B-cells. A novel CD37SMIP was previously demonstrated to mediate superior direct apoptosis and NK-cell mediated killing of chronic lymphocytic leukemia (CLL) and other B-cell malignancies. Methods: Given the superior in vitro apoptosis observed with CD37SMIP treatment and early clinical activity observed in highly refractory CLL patients, we hypothesized that a unique mechanism of cell killing was utilized by CD37SMIP. This was pursued in preclinical studies outlined below. Results: Unlike many other agents utilized to treat CLL, death mediated by CD37SMIP does not depend upon caspase activation. Nonetheless, CD37SMIP treatment of CLL cells promotes time-dependent induction of mitochondrial membrane depolarization, mitochondrial translocation of Bax, and up-regulation of Bim protein. CD37SMIP Bim protein induction occurred concomitantly with an increase in BIM mRNA levels. Electrophoretic mobility shift assay using oligonucleotides of the BIM promoter demonstrated increased protein binding activity in nuclear extracts derived from CD37SMIP treated cells and the physical interaction of FoxO3a transcription factor with the FoxO3a responsive element in the BIM promoter was demonstrated using a “protein pull down” assay and confirmed by chromatin immunoprecipitation assays. Furthermore, CD37SMIP treatment significantly increased BIM promoter regulated luciferase reporter expression in B-CLL cells. Consistent with a primary role of Bim up-regulation in mitochondrial membrane destabilization and apoptosis, transfection of CLL cells with BIM siRNA resulted in inhibition of CD37SMIP-induced mitochondrial membrane depolarization and apoptosis. Conclusions: These studies demonstrate CD37SMIP mediated apoptosis in CLL cells occurs via FoxO3a-dependent transcriptional up-regulation of BIM protein. This distinct mechanism of apoptosis utilized by CD37SMIP contrasts it with other agents used for CLL treatment. Additionally, it provides a mechanism for the promising clinical activity of TRU-016 (humanized CD37SMIP) observed to date in refractory CLL patients. [Table: see text]

We used primary mouse corneal epithelial cells (pMCE) to examine the role of Kcnj10 in determining membrane K+ conductance and cell membrane potential and in regulating EGF/TGFA release. Western blot, immunostaining, and RT-PCR detected the expression of Kcnj10 in mouse cornea. The single channel recording identified the 20-pS inwardly rectifying K+ channels in pMCE of WT mice, but these channels were absent in Kcnj10 −/−. Moreover, the whole cell recording demonstrates that deletion of Kcnj10 largely abolished the inward K+ currents and depolarized the cell membrane K+ reversal potential (an index of the cell membrane potential). This suggests that Kcnj10 is a main contributor to the cell K+ conductance and it is pivotal in generating membrane potential in cornea. Furthermore, to test the hypothesis that Kcnj10 expression plays a key role in the stimulation of growth factors release, we employed an immortalized human corneal epithelial cell line (HCE) transfected with siRNA-Kcnj10 or siRNA-control. Levels of TGFA and EGF secreted in the medium were measured by ELISA. Coimmunoprecipitation, biotinylation, and pull-down assay were used to examine the expression of EGFR and the GTP bound form of Rac1 (active Rac1). Downregulation of Kcnj10 activated Rac1 and enhanced EGF/TGFA release, which further contributed to the upregulation of EGFR phosphorylation and surface expression. We conclude that Kcnj10 is a main K+ channel expressed in corneal epithelial cells and the inhibition of Kcnj10 resulted in depolarization, which in turn induced an EGF-like effect.

The multidrug resistance (MDR) phenotype is one of the major obstacles in the treatment of chronic myeloid leukemia (CML) in advantage stages such as blast crisis. In this scenario, more patients develop resistance mechanisms during the course of the disease, making tyrosine kinase inhibitors (TKIs) target therapies ineffective. Therefore, the aim of the study was to examine the pharmacological role of CNN1, a para-naphthoquinone, in a leukemia multidrug resistant cell line. First, the in vitro cytotoxic activity of Imatinib Mesylate (IM) in K-562 and FEPS cell lines was evaluated. Subsequently, membrane integrity and mitochondrial membrane potential assays were performed to assess the cytotoxic effects of CNN1 in K-562 and FEPS cell lines, followed by cell cycle, alkaline comet assay and annexin V-Alexa Fluor® 488/propidium iodide assays (Annexin/PI) using flow cytometry. RT-qPCR was used to evaluate the H2AFX gene expression. The results demonstrate that CNN1 was able to induce apoptosis, cell membrane rupture and mitochondrial membrane depolarization in leukemia cell lines. In addition, CNN1 also induced genotoxic effects and caused DNA fragmentation, cell cycle arrest at the G2/M phase in leukemia cells. No genotoxicity was observed on peripheral blood mononuclear cells (PBMC). Additionally, CNN1 increased mRNA levels of H2AFX. Therefore, CNN1 presented anticancer properties against leukemia multidrug resistant cell line being a potential anticancer agent for the treatment of resistant CML.

The study of ion channel-mediated changes in membrane potential using the conventional bisoxonol fluorescent dye DiBAC4(3) has several limitations, including a slow onset of response and multistep preparation, that limit both the fidelity of the results and the throughput of membrane potential assays. Here, we report the characterization of the FLIPR Membrane Potential Assay Kit (FMP) in cells expressing voltage- and ligand-gated ion channels. The steady-state and kinetics fluorescence properties of FMP were compared with those of DiBAC4(3), using both FLIPR and whole-cell patch-clamp recording. Our experiments with the voltage-gated K+ channel, hElk-1, revealed that FMP was 14-fold faster than DiBAC4(3) in response to depolarization. On addition of 60 mM KCl, the kinetics of fluorescence changes of FMP using FLIPR were identical to those observed in the electrophysiological studies using whole-cell current clamp. In addition, KCl concentration-dependent increases in FMP fluorescence correlated with the changes of membrane potential recorded in whole-cell patch clamp. In studies examining vanilloid receptor-1, a ligand-gated nonselective cation channel, FMP was superior to DiBAC4(3) with respect to both kinetics and amplitude of capsaicin-induced fluorescence changes. FMP has also been used to measure the activation of KATP1 and hERG.2 Thus this novel membrane potential dye represents a powerful tool for developing high-throughput screening assays for ion channels.

Abstract The anti-neutrophil mAb PMN 7C3 and IIC4 inhibited the respiratory burst of neutrophils as measured by the generation of superoxide anion or hydrogen peroxide in response to PMA, serum-treated zymosan, and FMLP. To examine the effect of these mAb on neutrophil transmembrane potential, a fluorescent probe was used in a continuous assay. Compared with control cells, antibody-treated neutrophils were partially depolarized at rest and had a blunted response when stimulated. The F(ab)2 fragment of PMN 7C3 had similar effects on both the respiratory burst and transmembrane potential, whereas the Fab fragment did not. The unrelated antineutrophil mAb 31D8 had no effect on either the respiratory burst or on transmembrane potential. Neutrophils suspended in high potassium buffers also exhibited partial depolarization of the resting cell membrane and a blunted depolarization response to stimuli and produced less superoxide anion and hydrogen peroxide in response to stimuli than did control cells in physiologic buffer. Exposure of neutrophils to 2-deoxy-D-glucose resulted in dose- and time-dependent depression of the respiratory burst. 2-Deoxy-D-glucose also caused depolarization of the resting membrane and impaired subsequent stimulus-induced depolarization. Similar effects were seen with addition of iodoacetamide or depletion of glucose. The parallel effects of anti-neutrophil mAb, depolarizing buffers, and glycolytic inhibitors on both neutrophil membrane depolarization and activation of the respiratory burst indicate a close association between these two events. The evidence suggests that the inhibitory effects of these antibodies are mediated through partial membrane depolarization which interferes with signal transduction on subsequent stimulation of the cells. The impairment in oxidative responses to phorbol esters as well as to receptor-dependent activating agents points to interruption at a distal step, e.g., subsequent to Ca2+ mobilization.

Two oxovanadium(IV) complexes of [VO(msatsc)(phen)], (1) (msatsc = methoxylsalicylaldehyde thiosemicarbazone, phen = phenanthroline) and its novel derivative [VO (4-chlorosatsc)(phen)], (2) (4-chlorosatsc = 4-chlorosalicylaldehyde thiosemicarbazone), have been synthesized and characterized by elemental analysis, IR, ES-MS,1H NMR, and magnetic susceptibility measurements. Their antitumor effects on BEL-7402, HUH-7, and HepG2 cells were studied by MTT assay. The antitumor biological mechanism of these two complexes was studied in BEL-7402 cells by cell cycle analysis, Hoechst 33342 staining, Annexin V-FITC/PI assay, and detection of mitochondrial membrane potential (ΔΨm). The results showed that the growth of cancer cells was inhibited significantly, and complexes1and2mainly caused in BEL-7402 cells G0/G1 cell cycle arrest and induced apoptosis. Both1and2decreased significantly the ΔΨm, causing the depolarization of the mitochondrial membrane. Complex2showed greater antitumor efficiency than that of complex1.

SCOPO: 1) valutare l’espressione della proteine che regola il trasporto del cloro transmembrana (CFTR) e la sua attività funzionale nei monociti; 2) creare delle linee cellulari immortalizzate a partire da linfociti B aventi genotipi differenti; 3) valutare linee cellulari immortalizzate. CONOSCENZE DI BASE: La Fibrosi Cistica (CF), la più commune e grave malattia autosomale diffusa nei Paesi Caucasici, è dovuta alla presenza di mutazioni sul gene CFTR. Sebbene la CF sia una malattia multi organo, la patologia polmonare è la principale causa di morte tra i pazienti CF. E’ caratterizzata da una infiammazione cronica, conseguenza di un’infezione batterica. La suscettibilità alle infezioni batteriche non è completamente conosciuta, anche se il coinvolgimento di CFTR nelle funzioni microbicide dei macrofagi sta emergendo in questo periodo. I macrofagi differenziano in situ a partire dai monociti infiltati nel tessuto e mostrano una marcata variabilità morfologica pur avendo comuni funzioni cellulari e molecolari. Sebbene l’espressione di CFTR nei macrofagi alveolari sia stata descritta, la sua espressione nei monociti non è ancora stata riportata anche se queste cellule risultano essere più accessibili per studi funzionali e di espressione. La valutazione dell’espressione e della funzione del CFTR nelle cellule mononucleari del sangue periferico (PBMC) è un pre-requisito per valutare il loro ruolo ed il loro potenziale utilizzo in diagnostica e nello sviluppo di nuovi farmaci con azione sul difetto molecolare del CFTR. METODI: Purificazione dei monociti e dei linfociti B da sangue intero; produzione del virus Epstein-Barr (EBV); immortalizzazione dei linfociti B mediante EBV; isolamento RNA e analisi dell’mRNA mediante PCR; Real-time PCR; Western blotting; citometria di flusso; immunofluorescenza; depolarizzazione di membrana; misurazione delle differenze dei potenziali nasali; analisi dei dati. RISULTATI: Utilizzando un anticorpo anti-CFTR policlonale e due monoclonali che riconoscono differenti epitopi, abbiamo rilevato mediante western blotting tutte le forme conosciute del CFTR. La citometria di flusso e la microscopia confocale ha confermato l’espressione di CFTR e la sua localizzazione su membrana. Abbiamo osservato che i monociti non-CF, dopo stimolazione con uno specifico agonista di CFTR, mostravano un aumento dell’intensità di fluorescenza, una variazione che non abbiamo rilevato nei monociti CF. Questi risultati hanno suggerito una correlazione dell’attività di CFTR con la depolarizzazione della membrana ed i dati sono stati confermati tramite uno specifico inibitore, CFTR (inh)-172. Questo approccio è stato comparato alla misurazione dei potenziali nasali (NPD) eseguiti sugli stessi soggetti ed la sovrapposizione dei dati ha rilevato una forte corrispondenza tra le due tecniche. I linfociti B sono stati immortalizzati mediante EBV e sono stati utilizzati come potenziali modelli cellulari per valutare l’attività del CFTR. Abbiamo rilevato la maggiore forma glicosilata di CFTR in queste linee immortalizzate utilizzando un anticorpo monoclonale anti-CFTR. Una forma a minore peso molecolare è stata anch’essa evidenziata mediante questo anticorpo ed uno policlonale. La citometria di flusso e la microscopia confocale ci hanno permesso di confermare l’espressione e la localizzazione su membrana del CFTR. Il test di depolarizzazione della membrana è stato applicato sulle cellule B immortalizzate ottenendo gli stessi risultati visti sui monociti. CONCLUSIONI: Abbiamo dimostrato l’espressione della proteina CFTR in monociti umani identificando una variante a peso molecolare corrispondente ad un basso livello di post-trasduzione della proteina. Questo è stato confermato utilizzando monociti con genotipo omozigote per la mutazione non-sense i quali perdevano l’espressione della forma di CFTR. La citometria di flusso potrebbe essere utile per valutare l’espressione di CFTR. Infatti abbiamo dimostrato che può distinguere tra non-CF ed eterozigoti da pazienti CF mediante la marcatura CD14/Rb-AF488 dei monociti. L’analisi della depolarizzazione di membrana su singola cellula ha confermato l’espressione funzionale del CFTR mostrando una elevata depolarizzazione di membrana a seguito della stimolazione delle cellule con uno specifico agonista del CFTR. Questo metodo potrebbe essere eseguito entro un paio di ore dal prelievo del sangue. Inoltre, è facilmente riproducibile con un minimo disturbo e rischio per il paziente e potrebbe permettere una valutazione in corso d’opera degli effetti di alcune particolari terapie sull’espressione e sull’attività del CFTR. Abbiamo creato uno specifico indice capace di discriminare tra CF e non-CF. La sovrapposizione dei dati NPD e di quelli sull’attività funzionale del CFTR sui monociti risultata in una perfetta corrispondenza tra le due tecniche. Poiché NPD è un test diagnostico che si applica su soggetti con test del sudore dubbio o con almeno una mutazione non conosciuta, possiamo promuovere la valutazione dell’attività del CFTR nei monociti mediante tecniche ottiche come un utile metodo per valutare l’attività di CFTR per la ricerca, includendo lo sviluppo di nuovi farmaci e la diagnosi. Dato che le cellule primarie hanno disponibilità limitata in termini quantitativi, abbiamo preso vantaggio dall’osservazione che i linfociti esprimono CFTR. Le cellule B immortalizzate potrebbero essere utilizzate come modello cellulare per studiare l’espressione e l’attività del CFTR. Abbiamo rilevato l’espressione di una forma di CFTR che probabilmente rappresenta una isoforma processata a seguito dell’attività di una specifica calpaina nei linfociti come dimostrato in letteratura. Inoltre, l’indice ottenuto mediante lo studio della depolarizzazione di membrana ci ha permesso di discriminare tra gruppi CF e non-CF come osservato nei monociti. Tutti questi risultati hanno dimostrato che il CFTR è espresso ed è attivo nei linfociti umani e nelle cellule B immortalizzate. Per tale motivo, queste cellule possono essere sfruttate per valutare la risposta di specifiche mutazioni a nuovi farmaci diretti o indiretti sul difetto di base di CF.
AIM: 1) to evaluate the cystic fibrosis transmembrane conductance regulator (CFTR) protein expression and functional activity in monocytes; 2) to create immortalized cell lines from human B-lymphocyte cells characterized by different genotypes; 3) to evaluate CFTR protein expression in immortalized B cells. BACKGROUND: Cystic Fibrosis (CF), the most common autosomal severe disorder in Caucasians, is caused by mutations in the CFTR gene. Although CF is a multi-organ disease, the lung pathology is the main cause of morbidity and mortality of CF patients. It is characterized by chronic inflammation as a consequence of persistent bacterial infections by several opportunistic pathogens. Mechanisms leading to increased susceptibility to bacterial infections in CF are not completely known, although the involvement of CFTR in microbicidal functions of macrophages is emerging. Tissue macrophages differentiate in situ from infiltrating monocytes and display a remarkable variability in cell morphology although common molecular and cellular functions. Although expression of CFTR in alveolar macrophages has been described, its expression has not been reported in monocytes that are more accessible for expression studies and functional analysis tests than macrophages. Evaluation of expression and functional activity of CFTR in peripheral blood mononuclear cells (PBMC) is a pre-requisite to evaluate their role and their potential use in diagnostic and developing new drugs acting on the molecular defect of CF. METHODS: Purification of monocytes and lymphocyte B cells from whole blood; production of Epstein-Barr Virus (EBV); immortalization of Lymphocytes B cells by EBV; RNA isolation and CFTR mRNA analysis by reverse-transcription and polymerase chain reaction (PCR); quantitative real-time PCR (RT-qPCR); western Blotting; Flow cytometry assay; immunofluorescence; cell depolarization assay; Nasal Potential Differences (NPDs) assay; analysis of cell depolarization assay data. RESULTS: In this study western blotting using a polyclonal and two monoclonal anti-CFTR antibodies that recognize different epitopes detected all known forms of CFTR. Flow cytometry and confocal microscopy analysis confirmed expression of CFTR protein expression and its membrane localization. Increased fluorescence intensity, corresponding to membrane depolarization, was observed only when non-CF monocytes were stimulated with CFTR agonist, while CF monocytes did not show fluorescence variation. These results suggested a correlation between CFTR activity and membrane depolarization and data were confirmed using a specific CFTR inhibitor, CFTR (inh)-172. This approach was compared to NPD measurements performed in a subset of the same patients subjected to this analysis. Results obtained by NPD overlapped those obtained by the analysis of monocytes from non-CF donors and CF patients. B-lymphocytes were then immortalized by EBV and were tested as potential cell models for CFTR activity assays. The major glycosylated form of CFTR was detected in immortalized non-CF EVB-transformed B cell line by a monoclonal anti-CFTR antibody, but a band with minor molecular weight was also detected with this antibody and with a polyclonal anti-CFTR antibody. Flow cytometry and confocal assay allowed us to confirm CFTR expression and membrane location in these cell lines. Membrane depolarization test was applied in EBV-transformed B cells and the results confirmed a stimulus induced membrane depolarization in non- CF cells. CONCLUSION: We have demonstrated that CFTR proteins are expressed in human monocytes as a variant recognized by a specific antibody. Its molecular weight is consistent with a lower level of post-translational processing and its loss in patients carrying a homozygous non-sense mutation confirmed its presence in human monocytes. Flow cytometry could be also a useful method to evaluate CFTR expression. We demonstrated that it can distinguish between non-CF and HTZ subjects and CF patients analyzing stained CD14/Rb-AF488 monocytes. Single-cell membrane depolarization analysis confirmed that, upon stimulation with CFTR agonists, normal monocytes displayed a highly reproducible membrane depolarization activity consistent with the expression of functional CFTR. Single-cell depolarization assay could be performed within a few hours after blood collection. It is also easily repeatable with a minimal discomfort and risk for the patient and it could thus allow a time-course evaluation of effects of any particular therapy on CFTR expression or functional activity. A specific activity index was devised that appears capable to discriminate among CF and non-CF cells. Overlapping NPD data and functional activity data, we observed a perfect correspondence. Since NPD is a reference diagnostic test applied when a subject has borderline sweat test and at least one unidentified CFTR mutation, we might promote the evaluation of CFTR activity in monocytes by optical techniques as a useful tool to assess CFTR activity for basic and translational research, including drug development and diagnosis. As primary cells are available in limited amounts, we have taken advantage of the observation that CFTR-associated Cl- permeability has been demonstrated in lymphocytes. So, immortalized-B-cells could be useful as cellular model to study CFTR expression and activity. We observed a form of CFTR that likely represents a processed isoform possibly linked to specific calpain activity in lymphocyte cells as demonstrated in the literature. Furthermore, the index obtained by single-cell fluorescence imaging discriminated between non-CF and CF groups as shown in monocytes. All these results demonstrated that CFTR protein is expressed and is active in human lymphocytes and EBV-transformed B cells opening interesting perspectives in this field. Indeed, these cells can be exploited to evaluate the response of specific mutations to newly developed drugs acting directly or indirectly on the basic defect of CF.

Coagulation factor V (FV), is a single chain, multifunctional glycoprotein of Mr 350,000 which interacts with a variety of hemostatic proteins such as factor Xa, prothrombin, thrombin and protein C, on the surface of platelets and vascular endothelial cells. FV serves as both a cofactor and substrate in the generation of thrombin and plays a critical regulatory role in both physiologic hemostasis and pathologic thrombosis. The biosynthesis of FV and its subsequent expression are therefore expected to be precisely controlled and may differ in the three sites of synthesis - hepatocytes, endothelial cells, and megakaryocytes (MK). We have previously demonstrated that each guinea pig MK contains 500 times as much FV as in a platelet, as quantified by a competitive enzyme-linked-immunosorbent assay and expresses FV by cytoimmunofluorescence. De novo biosynthesis was demonstrated by incorporation of S-methionine into FV purified on a immunoaffinity column. The purified MK protein exhibited both FV coagulant activity and antigenicity. However, MK FV was more slowly activated by thrombin, more stable in the absence of Ca and exhibited a slightly higher M of 380,000 compared to plasma FV. Similar studies have documented biosynthesis in human MK. In addition, all morphologically recognizable MK enriched by elutriation from human bone marrow contained FV as documented by both monospecific polyclonal and monoclonal antibodies (MAb) to FV. All these cells bound FV since a murine MAb reacting with the light chain of FV (B38) labeled all cells. In contrast, 68% of cells synthesized FV since B10, a MAb to the activation peptide recognizing FV but not FVa, labeled this fraction. To determine whether immature nonnorphologically recognizable MK expressed FV, we identified these cells with an antiserum to human platelet glycoproteins and then probed them with B38. Seventy percent (70%) of such small cells expressed FV. In contrast, no small cells in MK colonies cloned in FV deficient medium expressed FV while only 40% of such colonies contained cells which expressed FV.To further probe the regulation of FV in MK we attempted to correlate the synthesis of FV as probed by MAb B10 with geometric mean cell diameter, stage and ploidy. No significant correlation of FV with any of these indicators of MK maturation. In contrast, preliminary studies suggest that low doses of tetradecanoyl phorbol acetate augment both the number of MK containing FV and the level of FV expressed by individual cells. Thus, FV synthesis may be regulated independent of size, stage, or ploidy and protein kinase C may play a role.To further define the molecular nature of FV in MK we found that purified FV was converted from a monomer to high Mr multimers by an enzyme derived from MK. These multimers resulting from covalent crosslinking since they were stable to SDS, 100° C and reducing agents. The responsible enzyme appeared to be MK FXIIIa since it required C, was inhibited by agents which react with the active site thiol group and was blocked by pseudoamine donor substrates such as putrescine. In addition, FXIIIa was directly demonstrated in guinea pig MK by a specific activity stain. Other investigators have established that FV became irreversibly associated with platelet cytoskeletons after exposure to thrombin. tested whether FXIIIa might mediate this association by performing ligand blotting of platelet membrane proteins using 125I-FV(FV*). Only actin of all the membrane proteins was detected by radioautography. The binding of FV* to the cytoskeleton was dependent in the presence of Ca and FXIIIa. In purified systems crosslinked complexes containing FV* or radiolabeled actin were detected in separate experiments. In whole platelets, the formation of the heteropolymer, after thrombin stimulation, was inhibited by antibodies to FXIII a chain, FV activation peptide (B10) or actin. Endogenous platelet FV was also dependent on FXIII for incorporation into the platelet cytoskeleton after thrombin stimulation. When thrombin-treated FV was crosslinked to actin only the activation peptide (150 kDa) was crosslinked. The light chain or heavy chain of FVa were not involved. Thus FXIIIa play an important role in the binding of FV in platelets to the cytoskeleton during activation and secretion.Further studies of FV in megakaryocytes are necessary to define the regulation of biosynthesis and the control of expression which dictate its critical role in hemostasis and thrombosis.