Search

Culicoides (Diptera: Ceratopogonidae) serve as vectors of several mammalian and avian diseases, including bluetongue, Schmallenberg, African horse sickness, avian malaria and Oropouche. Host preference investigations are necessary to assess the transmission routes of vector-borne diseases and to inform mitigation strategies. A recent study examining the main sensory structures (palps and antennae) of Culicoides species suggests that they be classified as ornithophilic or mammalophilic according to their feeding habits. We analyzed Culicoides host preferences according to the literature and carried out a multiple correspondence analysis linking these preferences with morphological data. Seven out of 12 variables were found to be reliable predictors of host preference in Culicoides species: Antenna Flagellomer-Sensilla Coeloconica-Number: (7-10) and (11-13); Antenna Flagellomer-Sensilla Coeloconica IV-X: presence; Palpus-size: wide and/or narrow opening and shallow pit; Palpus-Shape: strongly swollen; Antenna-Short sensilla trichodea-distal part segment IV to X-Number: 2 seta each. Our results demonstrate that the presence of sensilla coeloconica and the maxillary palpus can be used to separate ornithophilic and mammalophilic or ornithophilic/mammalophilic species., Competing Interests: The authors declare there are no competing interests.

An ideal multiphoton fluorescent nanoprobe should combine a nanocrystal with the largest possible two-photon absorption cross section (TPACS) and the smallest highly specific recognition molecules bound in an oriented manner. CdSe/ZnS quantum dots (QDs) conjugated to 13-kDa single-domain antibodies (sdAbs) derived from camelid IgG or streptavidin have been used as efficient two-photon excitation (TPE) probes for carcinoembryonic antigen (CEA) imaging on normal human appendix and colon carcinoma tissue. The TPACS for some conjugates was higher than 49,000 GM (Goeppert-Mayer units), considerably exceeding that of organic dyes being close to the theoretical value of 50,000 GM calculated for CdSe QDs. The ratio of sdAb-QD emission to the autofluorescence for 800 nm TPE was 40 times higher than that for 457.9 nm one-photon excitation. TPE ensures a clear discrimination of CEA-overexpressing tumor areas from normal tissue. Oriented sdAb-QD conjugates are bright specific labels for detecting low concentrations of antigens using multiphoton microscopy., From the Clinical Editor: This study demonstrates carcinoembryonic antigen imaging on normal human appendix and colon carcinoma tissue utilizing CdSe/ZnS quantum dots conjugated to streptavidin or to 13-kDa single-domain antibodies as efficient two-photon excitation probes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Common strategy for diagnostics with quantum dots (QDs) utilizes the specificity of monoclonal antibodies (mAbs) for targeting. However QD-mAbs conjugates are not always well-suited for this purpose because of their large size. Here, we engineered ultrasmall nanoprobes through oriented conjugation of QDs with 13-kDa single-domain antibodies (sdAbs) derived from llama IgG. Monomeric sdAbs are 12 times smaller than mAbs and demonstrate excellent capacity for refolding. sdAbs were tagged with QDs through an additional cysteine residue integrated within the C terminal of the sdAb. This approach allowed us to develop sdAbs-QD nanoprobes comprising four copies of sdAbs coupled with a QD in a highly oriented manner. sdAbs-QD conjugates specific to carcinoembryonic antigen (CEA) demonstrated excellent specificity of flow cytometry quantitative discrimination of CEA-positive and CEA-negative tumor cells. Moreover, the immunohistochemical labeling of biopsy samples was found to be comparable or even superior to the quality obtained with gold standard protocols of anatomopathology practice. sdAbs-QD-oriented conjugates as developed represent a new generation of ultrasmall diagnostic probes for applications in high-throughput diagnostic platforms., From the Clinical Editor: The authors report the development of sdAbs-QD-oriented conjugates, comprised of single domain antibodies that are 12 times smaller than regular mAb-s and quantum dots. These ultrasmall diagnostic probes represent a new generation of functionalized ODs for applications in high-throughput diagnostic platforms., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Semiconductor quantum dots (QDs) are proved to be unique fluorescent labels providing excellent possibilities for high-throughput detection and diagnostics. To explore in full QDs' advantages in brightness, photostability, large Stokes shift, and tunability by size fluorescence emission, they should be rendered stable in biological fluids and tagged with the target-specific capture molecules. Ideal QD-based nanoprobes should not exceed 15nm in diameter and should contain on their surface multiple copies of homogeneously oriented highly active affinity molecules, for example, antibodies (Abs). Direct conjugation of QDs with the Abs through cross-linking of QDs' amines with the sulfhydryl groups issued from the reduced Abs' disulfide bonds is the common technique. However, this procedure often generates conjugates in which the number of functionally active Abs on the surface of QDs does not always conform to expectations and is often low. Here we have developed an advanced procedure with the optimized critical steps of Ab reduction, affinity purification, and QD-Ab conjugation. We succeeded in reducing the Abs in such a way that the reduction reaction yields highly functional, partially cleaved, 75-kDa heavy-light Ab fragments. Affinity purification of these Ab fragments followed by their tagging with the QDs generates QD-Ab conjugates with largely improved functionality compared with those produced according to the standard procedures. The developed approach can be extended to conjugation of any type of Ab with different semiconductor, noble metal, or magnetic nanocrystals., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: To evaluate the local tissue concentrations of the antineoplastic agent doxorubicin and the amount of drug still present inside drug delivery embolization beads at different time points after embolization and to compare doxorubicin levels with histologic modifications around the beads in a pig liver model. It was hypothesized that doxorubicin-eluting beads maintain cytotoxic concentrations of drug locally over a period of several weeks, as suggested by in vitro elution tests., Materials and Methods: Left lobe hepatic artery embolization was performed in 10 pigs with 100-300-microm or 700-900-microm beads loaded with 37.5 mg doxorubicin/mL. Control unloaded 100-300-microm beads were injected in five pigs. Livers were sampled 28 days or 90 days after embolization. The amount of drug retained inside the beads was assessed with infrared microspectroscopy. Doxorubicin concentration and distribution in the tissue around the beads were determined with microspectrofluorimetry and compared with tissue modifications on hematein eosin saffron-stained sections., Results: Doxorubicin-eluting beads eluted 43% of their initial drug load after 28 days and 89% after 90 days. Doxorubicin was present in tissues around the beads at both time points, with a significant decrease over time (P = .0004). The drug was detected at distances as far as 600 microm from the bead edge. Doxorubicin tissue concentrations ranged from 0.55 microM to 6.80 microM, [corrected] which are cytotoxic levels in hepatocyte cell cultures. High concentrations of drug were associated with coagulative necrosis of liver parenchyma. Doxorubicin-eluting beads 100-300 microm in size induced more necrosis than 700-900-microm beads (P = .0036)., Conclusions: Doxorubicin-eluting beads deliver high concentrations of the drug over a period of at least 3 months at several hundred micrometers from the bead, leading to significant cytotoxic effects., (Copyright (c) 2010 SIR. Published by Elsevier Inc. All rights reserved.)

Diffusion-time distribution analysis (DDA) has been used to explore the plasma membrane fluidity of multidrug-resistant cancer cells (LR73 carcinoma cells) and also to characterize the influence of various membrane agents present in the extracellular medium. DDA is a recent single-molecule technique, based on fluorescence correlation spectroscopy (FCS), well suited to retrieve local organization of cell membrane. The method was conducted on a large number of living cells, which enabled us to get a detailed overview of plasma membrane microviscosity, and plasma membrane micro-organization, between the cells of the same line. Thus, we clearly reveal the higher heterogeneity of plasma membrane in multidrug-resistant cancer cells in comparison with the nonresistant ones (denoted sensitive cells). We also display distinct modifications related to a membrane fluidity modulator, benzyl alcohol, and two revertants of multidrug resistance, verapamil and cyclosporin-A. A relation between the distribution of the diffusion-time values and the modification of membrane lateral heterogeneities is proposed.

In continuation with our previous study using fluorescein-isothiocyanate (FITC)-Lys-Arg-Phe-Lys (KRFK) peptide, the aim of this work was to study the interaction of the unlabelled KRFK with calcium alginate gel microspheres coated with a serum albumin (HSA)-alginate membrane prepared using a transacylation method. Coated microspheres were prepared with two main sizes and two gel strengths. Control microspheres made of cross-linked alginate-HSA without calcium alginate gel were also prepared. A series of loading and release assays conducted with methylene blue showed the requirement of inner gel for binding the cationic molecule. Release experiments were performed in different media using unlabelled KRFK and coated microspheres. A plateau was reached within 1h, in contrast with the slow release of the FITC-peptide observed in our previous work. This discrepancy was attributed to modified properties of the labelled peptide. Adsorption assays of KRFK on coated microspheres were performed in the presence of growing concentrations of NaCl or imidazole. The ions were able to displace the peptide from the particles, which demonstrated ionic interactions, probably involving carboxylate groups of alginate. Adsorption isotherms showed that gel strength influenced affinity (4x10(5) L/mol or 8x10(5) L/mol for gelation with 5% or 20% CaCl(2), respectively). Binding site number doubled (from 2.6x10(-7) mol/mg to more than 5x10(-7) mol/mg) when microsphere size decreased from 450 microm to 100 microm. Binding sites were assumed to be located in the gel underneath the membrane.

In solid tumors, the cell microenvironment appears to be a key determinant in the emergence of drug resistance, a major obstacle to the successful use of antitumor drugs. Our aim was to determine whether type I collagen and fibronectin, proteins of the extracellular matrix, were able to influence the antimigratory properties induced by the antitumor drug doxorubicin. These properties were investigated at doxorubicin concentrations of 10 and 20 nM, which do not affect cell proliferation on a 24 h drug exposure. Using videomicroscopy, we found that these subtoxic doses of doxorubicin were sufficient to inhibit individual tumor cell motion on two-dimensional plastic surfaces. Such a drug treatment induced a dramatic disturbance of actin stress fiber formation and of vinculin distribution in 80% of cells. In contrast, on extracellular matrix proteins, cell speed was unaffected by drug and perturbation of both actin network and vinculin distribution was detected in only 50% of cells, suggesting a protective effect of the microenvironment. In addition, the phosphorylation of focal adhesion kinase and GTPase RhoA was less affected by doxorubicin with cells cultured on extracellular matrix proteins. In conclusion, our findings indicate that the cell microenvironment prevents drug-dependent inhibition of cell migration in vitro. They reveal cell locomotion as a key factor of microenvironment-mediated drug resistance. This new concept needs to be exploited in in vitro models to optimize the screening of new antimigratory drugs.

1alpha,25-dihydroxyvitamin D3 (VD3) and the EB1089 analog are well known for their roles in the modulation of proliferation and the differentiation of several malignant cells. In addition, VD3 or EB1089 displayed a high disposal of oxidant features and the ability to cause release of reactive oxygen species (ROS). We attempted to enhance HL60 cell differentiation and to limit ROS generation, by the association of deltanoids with doxorubicin and the antioxidants catalase (CAT), superoxide dismutase (SOD) and N-acetyl cystein (NAC). Differentiation of HL60 cells into monocytic lineage was studied by expression of mRNA, protein CD14 and functional differentiation by the nitroblue tetrazolium assay. The 2',7'-dichlorodihydrofluorescein diacetate (H2-DCFDA) dye allowed to evaluate in situ ROS generation. When associated with 0.1 nM EB1089, 15 nM doxorubicin induced an increase of differentiated cell percentage from 29% to 87% and did not affect VD3-treated cells. The association with doxorubicin also induced a significant increase of ROS release (p<0.05) versus VD3 and EB1089-treated cells. These results correspond to additivity of individual effects of doxorubicin and deltanoids. Antioxidant agents (10 nM NAC, 50 U/ml SOD or 2000 U/ml CAT) were associated with 10 nM VD3 or 1 nM EB1089 for 72 h. Compared to VD3 and EB1089 treatments, associations with antioxidants induced a slight increase of differentiated cells and a significant increase of CD14 mRNA. The highest differentiation effect occurred in the case of the EB1089-NAC association. Antioxidants induced a decrease (p<0.05) in ROS release generated by VD3 or EB1089 near the level of untreated cells. Thus, antioxidant agents demonstrated a protective effect against VD3 and EB1089 oxidative cytotoxicity and an enhancement of the monocyte differentiation. Combinations of antioxidants with deltanoids could dissociate the oxidative stress and differentiation.

Expression of three major resistance genes MDR1, MRP1 and LRP was investigated in small cell lung cancer, non-small cell lung cancer and metastasis. Single biopsies of bronchoscopy from 73 patients were performed to investigate expression of these three resistance genes by reverse transcriptase-polymerase chain reaction. Relations between gene expression and patient age, smoking status, histology, and chemotherapy were evaluated. A more frequent expression of MDR1 (77 versus 66%), MRP1 (91 versus 72%) and LRP (77 versus 63%) genes was detected in the malignant biopsies than in the non-malignant, respectively. In the metastasis biopsies, expression of these genes was markedly increased. No significant difference was observed between specimens before and after chemotherapy. Biopsies from progressing cancer showed higher MDR1, MRP1 and LRP gene expression. In conclusion, these data reveal a major role of MRP1 in intrinsic resistance and the high gene expression of MDR1 and MRP1 in relapsed diseases.

The binding and the diffusion of mitoxantrone (MTX) through the plasma membrane was performed by Förster resonance energy transfer (FRET) from the membrane fluorescent donor (4Di-10ASP) to the co-localized acceptor MTX. The MTX addition to living 4Di-10ASP-tagged cells resulted in the rapid quenching of the probe emission (1s), revealing the MTX binding to the outer leaflet. Then, a slower quenching (about 90s) occurred which corresponded to the MTX flip-flop into the inner leaflet. Changes of MTX integration into the plasma membrane were described in BCRP-overexpressed cells (HCT-116R) treated with (i) the BCRP inhibitor fumitremorgin C (FTC), (ii) cyclosporin A (CSA) and (iii) benzyl alcohol (BA). Treatments with FTC or CSA showed 80% and 40% higher flip-flop of MTX from the outer to the inner leaflet of HCT-116R cells. The addition of BA clearly increased the MTX integration into both outer and inner leaflets. Confocal fluorescence microscopy displayed that FTC, CSA and BA enhanced MTX accumulation in HCT-116R. In conclusion, Fumitremorgin C and agents modulating MTX accumulation resulted in higher MTX integration in the resistant cell membrane and could disrupt the membrane cohesion. This energy transfer method appears well-adapted to describe the drug diffusion through the plasma membrane of living cells.

In solid tumors, chemotherapeutics must adequately diffuse through the extracellular compartment to achieve their cytotoxic effect. Using quantitative microspectrofluorometry, both Doxorubicin penetration through three-dimensional (3D) collagen I matrices and its subsequent intranuclear accumulation into HT-1080 cells cultured in this microenvironment were directly assessed. Evidence that collagen delayed the Doxorubicin penetration for 1 h is presented. During that period, drug concentrations were lower in the nuclei in 3D compared to 2D matrices. Anthracyclines were also found to exhibit similar cytotoxicity in 2D and 3D after long term incubation. In conclusion, in this 3D culture model, collagen type I matrices delayed the early distribution of low molecular weight therapeutics and failed to affect their long-term cytotoxic effects, as previously reported. This model may provide a rationale for avoiding the emergence of intrinsic chemoresistance in tissue.

The ATP-binding cassette (ABC) transporters are one of the largest evolutionarily conserved families of proteins. They are characterized by the presence of nucleotide-binding domains (NBDs), which are highly conserved among organisms. In the present study, we used human and protozoan ABC sequences, and ATP-binding consensus motifs to screen the Toxoplasma gondii TwinScan2 predicted proteins database. We identified 24 ABC open reading frames (ORFs), whose deduced amino acid sequences exhibited all the typical biochemical features of the ABC family members. Fifteen of them clustered into five of the seven families of human ABC proteins: six ABCBs (drug, peptides and lipid export), two ABCCs (organic anion conjugates and drug export), one ABCE (Rnase L inhibitor, RLI, antibiotic resistance and translation regulation), one ABCF (drug resistance and regulation of gene expression) and five ABCGs (drug export and resistance). The nine other ORFs were represented by four ABCHs (energy-generating subunits), four SMCs (structural maintenance of chromosomes) and one member of unclear origin, whose closest homologue was the yeast Elf1 protein (mRNA export factor). A notable feature of the Toxoplasma ABC superfamily seems to be the absence of genes encoding ABCA and ABCD members. Expression analysis of ABC genes in tachyzoite and bradyzoite stages revealed the presence of ABC transcripts for all genes studied. Further research on the implication of these ABC proteins will increase our knowledge of the basic biology of Toxoplasma and provide the opportunity to identify novel therapeutic targets. To our knowledge, this is the first report of ABC transporters in T. gondii.

Chemoresistance remains the major obstacle to successful therapy of the lung cancer. The multi-drug resistance (MDR) is generally associated with altered expression of drug transporter proteins, such as P-glycoprotein (P-gp). So the distribution of P-gp on the membrane is of great importance to further study the interaction between drug and P-gp. In the present work, the P-gp of the H69/VP small-lung cancer cells was detected using monoclonal antibody UIC2. A secondary goat-anti mouse antibody coupled with biotin was used. The fluorescence emission was detected from a streptavidin-Texas Red. Results were investigated by a homemade scanning near-field optical microscope (SNOM) coupled to a confocal laser microspectrofluorometer (CLMF). Topographical images and localized spectra were obtained at the level of one cell membrane. It was found that the distribution of P-gp is not homogeneous and this observation is basically in accord with the fluorescent images obtained by classical microscopy. The distribution of P-gp would be localized in a higher region on a cell surface. This methodology would also enhance our understanding of MDR under physiological conditions.

A selective analysis of adsorbed mitoxantrone (MTX) was performed by surface-enhanced Raman scattering (SERS) at the range of cellular membrane. Disruption of the membrane fluidity was carried out to appraise changes in membrane adsorption of MTX and drug uptake in sensitive (HCT-116 S) and resistant BCRP/MXR (HCT-116 R) cells. Based on spectral MTX modifications, micro-SERS spectroscopy discriminated clearly drug adsorption phenomena on plasma membrane from drug in solution. A 3-fold higher SERS intensity of MTX for HCT-116 R was observed concluding to a higher drug adsorption on resistant membrane. The increase of membrane fluidity with benzyl alcohol (BA) or chloroform (CF) resulted in a 3-fold decrease of MTX adsorption on HCT-116 R, exclusively. BA and CF improved intracellular accumulation of MTX (e.g., 823 and 191 pmol MTX/10(6) HCT-116 R incubated with or without BA). At 4 degrees C, drug accumulation measurements showed a decrease of MTX permeability in resistant membrane (42 pmol MTX/10(6) cells), restored with fluidizers (e.g., 342 pmol MTX/10(6) cells with BA). Fluorescence confocal microscopy involved an exclusive MTX emission around the plasma membrane of resistant cells whereas fluidizers increased the intracellular uptake of MTX in both cell lines at the same time with less drug emission around the plasma membrane. Changes of the membrane structure of resistant cells should modify both drug adsorption and membrane permeation.

We attempted to characterize the cellular autofluorescence phenomenon of living HL-60 cells and to appraise its modifications under oxidative stress conditions induced by 1 alpha,25(OH)(2)D(3) (VD(3)) and its analog EB1089. Autofluorescence emission spectra of human promyelocytic HL-60 leukemic cells were monitored using laser scanning confocal microspectrofluorometry under UV excitation. Evaluation of reactive oxygen species (ROS) release was performed using the 2',7'-dichlorodihydrofluorescein diacetate (H(2)-DCFDA) staining and fluorescence emission measurement. VD(3) (1, 10, 100 nM) or EB1089 (0.1, 1 and 10 nM) induces a decrease in autofluorescence emission intensity that can be attributed to the oxidation of the coenzyme nicotinamide adenine dinucleotide (phosphate) NAD(P)H into NAD(P)(+). A dose-dependent increase (p<0.05) in ROS release is observed in VD(3)- and EB1089-treated cells. As compared with VD(3)- or EB1089-treated cells, doxorubicin-VD(3) or doxorubicin-EB1089 treatments strongly decrease the autofluorescence intensity and induce a higher release of ROS (p<0.05). The association of antioxidants (N-acetyl cysteine, superoxide dismutase, catalase) with VD(3) or EB1089 induce a more limited autofluorescence decrease and a weaker ROS generation, as compared with VD(3) and EB1089 treated cells. In conclusion, the free radicals release, generated by VD(3) and EB1089, was associated with the decrease in autofluorescence emission and can be modulated by doxorubicin and antioxidants.

Surface-enhanced Raman scattering (SERS) spectroscopy was applied to analyze mitoxantrone (MTX) adsorption on the plasma membrane microenvironment of sensitive (HCT-116 S) or BCRP/MXR-type resistant (HCT-116 R) cells. The addition of silver colloid to MTX-treated cells revealed an enhanced Raman scattering of MTX. Addition of extracellular DNA induced a total extinction of MTX Raman intensity for both cell lines, which revealed an adsorption of MTX on plasma membrane. A threefold higher MTX Raman intensity was observed for HCT-116 R, suggesting a tight MTX adsorption in the plasma membrane microenvironment. Fluorescence confocal microscopy confirmed a relative MTX emission around plasma membrane for HCT-116 R. After 30 min at 4 degrees C, a threefold decrease of the MTX Raman scattering was observed for HCT-116 R, contrary to HCT-116 S. Permeation with benzyl alcohol revealed a threefold decrease of membrane MTX adsorption on HCT-116 R, exclusively. This additional MTX adsorption should correspond to the drug bound to an unstable site on the HCT-116 R membrane. This study showed that SERS spectroscopy could be a direct method to reveal drug adsorption to the membrane environment of living cells.

We examined xenobiotic transport and the effects of P-glycoprotein (Pgp) inhibitors on efflux function in Toxoplasma gondii tachyzoites. The fluorescence emission of JC-1 and daunorubicin (Pgp substrates) was determined in both extracellular tachyzoites and T. gondii-infected human KB cells. Dye accumulation and efflux were modulated by verapamil (Vp) and cyclosporin A (CsA), both of which are Pgp inhibitors. Red JC-1 emission was measured from 10(6) extracellular tachyzoites, using spectrofluorometry. The increase in red emission was significant from 1 microM concentration of both drugs and was higher with CsA than with Vp. Compared with untreated tachyzoites, JC-1 efflux was inhibited by 3 microM CsA and 3 microM Vp. With intracellular tachyzoites, the fluorescence distribution of daunorubicin (DNR) between the parasitophorous vacuole and the host cell was modulated by Vp and CsA. In media free of CsA and Vp, DNR emission inside intracellular tachyzoites was very weak, as observed by confocal microscopy. In the presence of CsA or Vp, DNR emission was markedly enhanced in tachyzoites but not in the whole vacuole. The modulation of DNR uptake seems to involve the tachyzoite membrane rather than the parasitophorous vacuole or host cell membranes. It suggests that Vp would inhibit the DNR efflux from intracellular tachyzoites through a transitory effect. In conclusion, these two Pgp inhibitors increase both extracellular and intracellular dye accumulation in living T. gondii, pointing to the existence of a transmembrane transport mediated by a Pgp homologue located on the parasite membrane complex.

Complexes formed between G-quadruplex (G4)-conformed oligonucleotides and four ethidium derivatives were studied by surface-enhanced Raman spectroscopy (SERS) to detail the topology of complexes that support a G4 stabilization. Ethidium bromide (EB), which presents a weak ability to stabilize oligonucleotides in G4 conformation, displayed no SERS intensity modification when bound to G4, as compared with the free EB. Three ethidium derivatives have been selected due to their higher ability to stabilize G4 than EB. Bound with G4-conformed oligonucleotides, SERS intensity of these three ethidiums decreased by factors of about 6, 3.5, and 15. The high SERS quenching was interpreted as a loss of accessibility of silver colloids for G4-bound ethidiums. This could represent a new selective parameter useful to identify G4-stabilizing molecules. To apraise the role of the oligonucleotide sequence on the interaction mode, complexes were formed with eight G4-conformed oligonucleotides in which the three loops were either 5'-TTA-3' or 5'-AAA-3'. Spectra of ethidiums were sensitive to both lateral loops, opposite to the 3' and 5' G4 ends. The sequence of these loops are believed to be selective in the interaction mode of ethidiums for G4.

A spectroscopic analysis of autofluorescence was investigated within the cell cytoplasm from cervical malpighian epithelia prepared on Thin-Prep smears. Autofluorescence emission spectra from 22 cervix were analyzed by microspectrofluorometry under a 363 nm laser excitation. Among the analyzed cervix, 6 were in normal limits, 6 in inflammatory limits, 5 were evocative of Low-Grade Squamous Intraepithelial Lesions (LGSILs) and 5 were evocative of High-Grade Squamous Intraepithelial Lesions (HGSILs). Cytoplasmic emission intensities at 450 nm of cells from inflammatory, LGSIL and HGSIL cervix were equivalent and were 3-fold higher than from normal cervix. All smears presented a two-fold lower autofluorescence emission in the cytoplasm than in the nucleus. The spectral profile analysis allows the discrimination of cells from inflammatory, LGSIL and HGSIL cervix. The 525/425 nm emission ratios were 0.75+/-0.1, 0.96+/-0.04 and 1.2+/-0.1 for inflammatory, LGSIL and HGSIL, respectively. We suggest that smears of normal, inflammatory, LGSIL and HGSIL cervix could be discriminated by the analysis of the 450 nm emission intensity and 525/425 nm emission ratios from cells of malpighian epithelia.

The presence of a nucleotide binding site on hsp90 was very controversial until x-ray structure of the hsp90 N-terminal domain, showing a nonconventional nucleotide binding site, appeared. A recent study suggested that the hsp90 C-terminal domain also binds ATP (Marcu, M. G., Chadli, A., Bouhouche, I., Catelli, M. G., and Neckers, L. M. (2000) J. Biol. Chem. 275, 37181-37186). In this paper, the interactions of ATP with native hsp90 and its recombinant N-terminal (positions 1-221) and C-terminal (positions 446-728) domains were studied by isothermal titration calorimetry, scanning differential calorimetry, and fluorescence spectroscopy. Results clearly demonstrate that hsp90 possesses a second ATP-binding site located on the C-terminal part of the protein. The association constant between this domain of hsp90 and ATP-Mg and a comparison with the binding constant on the full-length protein are reported for the first time. Secondary structure prediction revealed motifs compatible with a Rossmann fold in the C-terminal part of hsp90. It is proposed that this potential Rossmann fold may constitute the C-terminal ATP-binding site. This work also suggests allosteric interaction between N- and C-terminal domains of hsp90.

The effects of extracellular Mg2+ on both dynamic changes of [Ca2+]i and apoptosis rate were analysed. The consequences of spatial and temporal dynamic changes of intracellular Ca2+ on apoptosis, in thapsigargin- and the calcium-ionophore 4BrA23187-treated MCF7 cells were first determined. Both 4BrA23187 and thapsigargin induced an instant increase of intracellular Ca2+ concentrations ([Ca2+]i) which remained quite elevated (> 150 nM) and lasted for several hours. [Ca2+]i increases were equivalent in the cytosol and the nucleus. The treatments that induced apoptosis in MCF7 cells were systematically associated with high and sustained [Ca2+]i (150 nM) for several hours. The initial [Ca2+]i increase was not determinant in the events triggering apoptosis. Thapsigargin-mediated apoptosis and [Ca2+]i rise were abrogated when cells were pretreated with the calcium chelator BAPTA. The role of the extracellular Mg2+ concentration has been studied in thapsigargin treated cells. High (10 mM) extracellular Mg2+, caused an increase in basal [Mg2+]i from 0.8+/-0.3 to 1.6+/-0.5 mM. As compared to 1.4 mM extracellular Mg2+, 1 microM thapsigargin induces, in 10 mM Mg2+, a reduced percentage from 22 to 11% of fragmented nuclei, a lower sustained [Ca2+]i and a lower Ca2+ influx through the plasma membrane. In conclusion, the cell death induced by thapsigargin was dependent on high and sustained [Ca2+]i which was inhibited by high extracellular and intracellular Mg2+.

Image cytometry was applied to study the intracellular localization of autofluorescence and the influence of an oxidative stress on this emission. K562 erythroleukemia cancer cells were analyzed with a microspectrofluorometer, coupled with a Argon laser (Ar+) (363 nm). From each cell, 15 x 15 emission spectra were recorded in the 400-600 nm spectral range to generate a spectral image of autofluorescence. The intracellular locations of the autofluorescence emission and of the specific mitochondrial probe rhodamine 123 (R123) were matched. Under a 363 nm excitation, all spectra from K562 cells show equivalent profiles with a 455 nm maximum emission, near of reduced nicotinamide adenine dinucleotide-(Phosphate) solution (NAD(P)H) (465 nm maximum emission). The spatial distribution of autofluorescence is homogeneous and different from the one of R123. Hydrogen peroxide (H2O2) (200 microM) and menadione (Men) (5 microM) induce a weak spectral change and a decrease in autofluorescence intensity, down to 40% of the initial emission. Doxorubicin (Dox) induces a dose-dependent decrease in autofluorescence emission and a release of intracellular free radicals. When cells were pre-treated 1 h with 1 mM glutathione (GSH), Dox induces a lower free radicals release, no significant variation of autofluorescence intensity and a lower growth inhibitory effect. Images cytometry of autofluorescence suggest that the intracellular NAD(P)H would not be restricted to mitochondrial compartments. The release of free radicals was associated with a decrease in autofluorescence intensity, mainly attributed to NAD(P)H oxidation both inside and outside mitochondria.

Membrane potential changes in host cell plasma membrane were analyzed and the parasitophorous vacuole membrane (PVM) potential was characterized after infection by Toxoplasma gondii. Human monocytes infested by T. gondii were stained with two membrane potential sensitive dyes, DiOC(6)(3) carbocyanine and DiSBAC(2)(3) bis-oxonol, before fluorescence emission analysis by confocal laser scanning microscopy. After 24 and 48 h of infection, 34 and 39%, respectively, of monocytes showed several parasites (from two to six) per cell. At these infection times, significant decreases in cytoplasmic emissions were observed for both DiOC(6)(3) and DiSBAC(2)(3). Thus, hyperpolarisation of the host plasma membrane would occur consecutively to infection. Inside the parasitophorous vacuole, the fluorescence intensity of DiOC(6)(3) and DiSBAC(2)(3) increased significantly from 6 to 24 h after infection and the PVM became less polarised. Involvement of different ATPases in the membrane potential of infected monocytes was evaluated with ouabain, DCCD, omeprazole and sodium orthovanadate, ATPase inhibitors. All inhibitors induced a depolarisation of the plasma membrane. In the parasitophorous vacuole compartment, DCCD, omeprazole and sodium orthovanadate but not ouabain caused a significant depolarisation of the PVM, suggesting that H(+), H(+)/K(+) and P-type ATPases were at the origin of the PVM potential. This is the first report showing the presence of ion transporters in the T. gondii PVM and the existence of at least two members of the P-type family of ion pumps: an electrogenic H(+)ATPase and an electroneutral H(+)/K(+) ATPase.

Dietary surveys performed in Western countries show magnesium intakes lower than the recommended dietary allowances, suggesting a large prevalence of magnesium deficiency. Low brain magnesium as well as impaired magnesium metabolism have also been reported in various diseases such as migraine. To detect these deficiencies, a non-invasive and sensitive test assessing magnesium status is needed. Because magnesium is an intracellular cation, either total or ionized magnesium (Mg(2+)) of blood cells were suggested as the most adequate tests. Total magnesium levels in plasma, erythrocytes and lymphocytes and Mg(2+) in lymphocytes were analyzed in a group of 29 migraine patients and 18 control subjects. Results show significantly lower concentrations of total magnesium in erythrocytes (50.7+/-4.7 vs. 53.5+/-2.9 mg/l; P<0.01) and of Mg(2+) in lymphocytes (12.0+/-3.5 vs. 14.2+/-3.8 mg/l; P<0.05) in migraine patients as compared to controls. While a significant difference of mean values was noted between migraine patients and controls, an overlap of individual values was observed. These analyses were repeated on migraine patients before and after a 2-week intake of a mineral water containing 110 mg/l magnesium, and a significant increase in all intracellular magnesium concentrations with no effect on plasma magnesium was observed. These increased intracellular magnesium concentrations demonstrate the bioavailability of magnesium from this mineral water. Among the analyzed parameters, Mg(2+) in lymphocytes appears to be the most sensitive index of magnesium deficiency with a 15% decrease in migraine patients when compared to controls and a 16% increase after 2 weeks of a magnesium-rich mineral water intake.

We used confocal microspectrofluorometry to investigate intracellular distribution of pirarubicin or THP-DOX in parental K562, CEM, and LR73 tumor cells and their corresponding multidrug-resistant (MDR) strains. Each spectrum of a recorded image was considered as a combination of cell autofluorescence and fluorescence of the drug. In the cytoplasm of parental K562, CEM, and LR73 cells, THP-DOX fluorescence emission profile was similar to that of free drug in aqueous buffer. The (I550nm/I600nm) ratio was 0. 50 +/- 0.1. However, in the cytoplasm of resistant cells the 550-nm band profile was modified. The I550nm/I600nm ratio was 0.85 +/- 0.2 in MDR K562 cells, which is significantly different from the ratio in sensitive cells (p<0.01). This appeared first to correspond to accumulation and self-oligomerization of THP-DOX in cytoplasmic organelles of MDR cells. Transfection of LR73 cells with the mdr1 gene conferred this characteristic on the resistant LR73R cells. Bodipy-ceramide, a trans-Golgi probe, was co-localized with the typical fluorescence emission peak at 550 nm observed in the cytoplasm of MDR cells. This organelle has been shown to be more acidic in MDR cells. Moreover, this specific pattern was similar to that observed when anthracycline is complexed with sphingomyelin. The typical fluorescence emission peak at 550 nm decreased in MDR cells incubated simultaneously in the presence of the drug and quinine, verapamil, or S9788. Growth inhibitory effect and nuclear accumulation of THP-DOX data obtained on LR73R and LR73D cell lines showed that only during reversion of resistance by verapamil and S9788 was an increase of nuclear THP-DOX accumulation observed. Our data suggest that characteristics of molecular environment, such as higher pH gradient or lipid structures, would be potential mechanisms of multidrug-resistance via the sequestration of anthracyclines.

The kinetics of hydrolysis of the alpha-hydroxylactone ring of anticancer agents belonging to the camptothecin (CPT) series has been followed using their fluorescence emission. Data obtained for CPT, CPT-11, and SN-38, either in their free form or in the presence of DNA and/or topoisomerase I (top1), have been compared. DNA was modeled using three types of double-strand oligonucleotides corresponding to top1 cleavage site enhanced in the presence of the drug (olg1), top1 site independent of CPT (olg2), and nonspecific synthetic oligonucleotide containing only AT and no GC base pairs (olg3). Cleavage assays indicated the absence of top1-mediated cleavage on olg3, both in the presence and in the absence of CPT. The kinetics data also showed ratio-dependent stabilization of the lactone forms of CPTs when in the presence of an excess of olg1 or olg2, but not of olg3. These observations correlate with the previously reported preferential binding of CPTs to guanines. Although lactone hydrolysis was not perturbed by top1 alone, this enzyme hindered lactone stabilization by specific oligonucleotides. After addition of top1 to CPT-olg1 or CPT-olg2 complexes, the lactone ring of the drug was destabilized. No lactone stabilization was observed when olg1 was added to CPT-top1 complexes or when olg1-top1 complexes were added to CPT.

The effects of extracellular Mg2+ concentration have been investigated on the histamine-stimulated exocytotic process of human tracheal secretory gland (HTG) cells. The exocytosis of secretory granules (SG) was observed concomitantly with dynamic changes of intracellular Ca2+ ([Ca2+]i) and Mg2+ concentrations ([Mg2+]i). The rate of SG exocytosis was appraised by the decrease of quinacrine fluorescence emission. Dynamic changes of [Mg2+]i and [Ca2+]i in HTG cells were determined by the combined use of UV-microspectrofluorometry with Mag-Indo-1 and Indo-1 probes, respectively. High Mg2+ medium significantly inhibited the histamine-stimulated secretion. The influence of the extracellular and intracellular Mg2+ concentrations on [Ca2+]i was analyzed. Basal [Mg2+]i increased from 0.8 mM in a Mg(2+)-free medium to 1.7 mM in 10 mM Mg2+ medium. Histamine induced a [Mg2+]i increase which is dependent on extracellular Mg2+ concentration. The histamine stimulated [Ca2+]i rise was reduced in the presence of elevated Mg2+ extracellular medium and inhibitory effects of extracellular Mg2+ were concomitant with changes in [Mg2+]i. Our data suggest that the inhibition by extracellular Mg2+ of stimulated exocytosis is dependent on both the increase of [Mg2+]i and the inhibition of cytosolic Ca2+ influx.

Potent antitumor activity exhibited by 20-S-camptothecin (CPT) and numerous derivatives is known to be lost upon opening of the alpha-hydroxy-lactone ring of these drugs, hydrolyzable at neutral and basic pH. To quantify in 'real time' the lactone hydrolysis reaction in CPTs under physiological conditions, we have applied a non-perturbing approach by fluorescence spectroscopy. CPT and a set of its derivatives (21-lactam-S-CPT, 10,11-(methylenedioxy)-CPT, CPT-11, SN-38, topotecan, tricyclic ketone-CPT) with antitumor activity varying from negligible to 10 times that of CPT have been studied. Prior to the kinetic measurements, the effects of substitutions, pH, polarity of molecular environment, lactone ring opening (lactone-carboxylate transition) have been investigated in terms of the UV-visible absorption and fluorescence emission spectra of CPTs. Then the determined parameters of the fluorescence emission spectra corresponding to the respective lactone and carboxylate forms have been used to estimate the residual lactone percentage as a function of time. The reproducibility of the obtained data demonstrates that the spectroscopic approach provides a satisfactory precision for this kind of measurements. For CPT at pH 7.3, the lactone half-life was 29.4 +/- 1.7 min and the lactone percentage at equilibrium was 20.9 +/- 0.3%. Within a series of derivatives with substitutions at quinoline rings, the lactone half-life varied from 29 to 32 min and the equilibrium lactone content varied from 15% to 23%. For each compound, even slight increase of pH from 7.1 to 7.3 or from 7.3 to 7.6 logically leads to a remarkable decrease of both lactone half-life and equilibrium lactone percentage.

The molecular and ionic mechanisms responsible for the regulation of mucus exocytosis in human airway gland cells remain poorly defined. To determine whether dynamic changes of intracellular free Ca2+ concentration [Ca2+]i can promote different exocytotic responses, we monitored dynamic changes in [Ca2+]i and secretory granule (SG) exocytosis in individual human tracheal submucosal serous gland (HTG) cells. These changes were in response to exposure of the cells to three different secretagogues associated with airway inflammation and disease: human neutrophil elastase (HNE), histamine, and ATP. Dynamic changes in [Ca2+]i from single cells were determined with Indo-1/AM using quantitative UV laser microspectrofluorometry. The rate of SG exocytosis was measured in single cells by fluorescence videomicroscopy of SG degranulation and by the ELISA method. Exposure of HTG cells to a low concentration of HNE (1.0 microM) caused a high rate of SG exocytosis (52% decrease in the initial quinacrine fluorescence) during the first 8-min stimulation period compared with that observed following exposure of the cells to 100 microM histamine (10% decrease) or 100 microM ATP (6% decrease). In contrast to a rapid and transient rise in [Ca2+]i induced by histamine (1.0-100 microM) and ATP (10-100 microM), HNE (0.01-1 microM) generated asynchronous oscillations in [Ca2+]i over the first 8-min period. Depletion of internal Ca2+ stores with thapsigargin (500 nM) induced a significant reduction (P < 0.01) in the observed increases in [Ca2+]i upon addition of each of the secretagogues, but did not greatly affect the SG exocytotic responses. Interestingly, the removal of extracellular Ca2+ (+5 mM EGTA) significantly reduced (P < 0.01) both the [Ca2+]i increases and the rate of SG exocytosis following exposure to the secretagogues. We also demonstrate that the influx of extracellular Ca2+ and [Ca2+]i oscillations rather than the absolute level of [Ca2+]i regulate the rapid onset and extent of exocytotic responses to HNE in airway gland cells. Taken together, these results provide strong evidence that [Ca2+]i is a critical intracellular messenger in the regulation of exocytosis process in human airway gland cells.

To study the pH gradient status through membranes of acidic vesicles, either in sensitive or in multidrug-resistant living cancer cells, we monitored the fluorescence-emission spectra of acridine orange. Successive stainings with a pH-sensitive dye and AO showed that low-pH organelles were stained red by AO. In these compartments, high AO concentrations are driven by the pH gradient through membrane vesicles. The resulting rise in the dye's oligomeric/monomeric ratio induced an increase in the red/green (655-nm/530-nm) emission intensity ratio. Therefore, the accumulation of AO in acidic organelles was appraised by determination of the contribution of the red emission intensity (R%) in each emission spectrum, using laser scanning confocal microspectrofluorometry. In vesicles of multidrug-resistant K562-R cells, R% is significantly higher (72 +/- 10%) than the value (48 +/- 8%) from K562-sensitive cells (p < 0.001). This result is interpreted as a more important accumulation of AO in acidic cytoplasmic structures of resistant cells, which induces a shift from AO monomers (green emission) to self-associated structures (red emission). Equilibration of the pH gradient through acidic organelles was performed by addition of weak bases and carboxylic ionophores. Ammonium chloride (0.1 mM), methylamine (0.1 mM), monensine (10 microM), or nigericine (0.3 microM) all suppressed the initial difference of local AO accumulation between both cell lines. These agents decreased the red emission intensity for the resistant cell line but not for the sensitive one. The same effects were induced by 50 microM verapamil, a pleiotropic drug-resistance modulator. Our data allow the hypothesis of a higher pH gradient through membranes of acidic organelles, which would be a potential mechanism of multidrug resistance via the sequestration of weak bases inside these organelles.

Hyphenation of attenuated total reflection Fourier transform infrared spectroscopy and cluster analysis has been used to characterise a susceptible Escherichia coli K12 strain and the transconjugants TEM-1, TEM-2, TEM-3, SHV-2, SHV-3, SHV-4. A good discrimination of the susceptible strain from the transconjugants was obtained. Although a limited success was achieved in the differentiation of SHV and TEM phenotypes in general, results obtained with TEM-2 and SHV-3 were convincing. Spectral differences observed are ascribed to the global effects of the conjugation process, particularly their repercussions in the nucleic acids and carbohydrate absorbing regions, rather than to beta-lactamase point-mutations.

There is a large discrepancy between the changes in drug accumulation and the changes in drug cytotoxicity that accompany development of anthracycline in multidrug-resistant cells. Moreover, although different molecular targets for anthracyclines such as DNA, cell membranes, or enzymes like topoisomerases could be involved, mechanisms by which these compounds exert their cytotoxic and differentiating effects remain unclear. Studies of correlation between the biological effects of anthracyclines and drug uptake have given conflicting conclusions. For example, a decrease in drug cytotoxicity for different incubation temperatures has been observed in spite of the same intracellular anthracycline amount, suggesting that temperature-dependent cytotoxic effects may be mediated by drug interaction with the cell membrane. What we propose in this review are results of our laboratory which are in agreement with an action mechanism targeted to the nucleus. In fact, we have shown by using microspectrofluorometry, that identical nuclear anthracycline concentration induces the same degree of cytotoxicity, independent of cellular MDR phenotype and the anthracycline structure. Thus, we could acquire information on the mechanisms of drug resistance related to drug transport. We could also give evidence that this accumulation is increased when MDR modulators, such as verapamil and S9788 and cyclosporin A or anthracyclines are used. For clinical applications, our studies have already dealt with nuclear concentration measurements of doxorubicin in leukocytes of treated patients, and in vitro measurements of drug efflux from nuclei of acute leukemic cells and its correlation with P-glycoprotein expression. However, in these studies, there was no correlation between anthracycline nuclear accumulation in vitro and P-glycoprotein expression. In addition, from preliminary results, we have shown that some modulators such as quinine do not significantly increase nuclear accumulation of anthracyclines in MDR cells but are able to restore anthracycline sensitivity. Other authors have recently shown that quinine has a relatively weak effect on cellular doxorubicin accumulation in MDR cells but is able to completely restore doxorubicin sensitivity. They concluded that quinine has essentially intracellular targets involved in drug distribution (cytoplasm --> nucleus) from sequestration compartments. Our data contradict this and we believe that such modulator modifies the molecular environment of anthracyclines and/or their binding to a possible cytoplasmic target leading to different cell death. Thus, we conclude that mechanisms by which anthracyclines induce cell death, and ways by which chemotherapy fails in resistant cells remain complex and are related to more than one target.

The primary mechanism of imipenem resistance in Pseudomonas aeruginosa has been ascribed to an outer membrane impermeability owing to a loss of expression of protein D2. Attenuated total reflection-Fourier transform infrared spectroscopy in conjunction with statistical methods has been used as a new approach to rapidly discriminate four isogenic strains of P. aeruginosa--susceptible, less susceptible, and highly resistant to imipenem-- and to follow the structural modifications related to this low permeability. Decomposition of the broad protein and carbohydrate contours into underlying Gaussians and comparison of the susceptible and highly resistant strain provided quantitative and ultrastructural information on these strains. This methodology allows for discrimination not of the mutation itself but of its consequences observed in the protein and carbohydrate absorption regions. Its association with other existing biochemical methods may be envisaged since it may allow for rapid orientation of investigations in the field of bacterial resistance diagnosis.

The combined use of Mag-indo-1 probe and laser confocal UV-microspectrofluorometry allowed us to investigate the spatial and temporal dynamic changes of the Mg2+ variations in human tracheal gland (HTG) cells at the single cell level. Stimulation of HTG cells with either bradykinin, ouabain or extracellular high Mg2+ concentrations (up to 10 mM) induced increases in intracellular Mg2+ concentration [Mg2+]i. From a cytosolic basal concentration of 0.8 +/- 0.3 mM in a medium free of Mg2+, an increase in extracellular Mg2+ concentration from 1 to 10 mM, increased cytosolic [Mg2+]i from 1.4 +/- 0.6 to 1.8 +/- 0.8 mM after 10 min (p < 0.05). We also demonstrated using line-scanned spectral images within single cells, that the [Mg2+]i is distributed uniformally in the nucleoplasm, but in contrast, showed marked local differences among different cytoplasmic regions, thus suggesting a functional heterogeneity in the intracellular Mg2+ stores involved. The influx pathway for Mg2+ in HTG cells was not inhibited by verapamil and appeared to be independent of [Ca2+]i.

We measured intracellular free calcium concentrations ([Ca++]i) in the subcellular compartments of Toxoplasma gondii infected living cells using microspectrofluorometry and Indo-1 staining. [Ca++]i mapping was defined in infected and uninfected cells and in the neoformed parasitophorous vacuole (PV) 24 and 48 hr after parasite inoculation. At 24 hr after infection, a [Ca++]i gradient (PV/cytoplasm) was observed in favor of the PV in 72% of infected cells (p<0.001). Inside of the PV (lumen and parasites), [Ca++]i values appeared to be homogeneously distributed. At 48 hr after infection, the parasites had replicated and formed typical rosettes of more than 16 parasites. At this step, a positive [Ca++]i gradient (PV/cytoplasm) was detected in all analyzed cells (p<0.001). This result suggests that the PV (lumen and parasites) represents an individual subcellular compartment within the host cell that includes an independent [Ca++]i. Moreover, after 48 hr the cytoplasmic [Ca++]i decreased significantly (39 nM) compared with that measured from uninfected cells (53 nM) (p <0.05). Furthermore, the exit of Toxoplasma mediated by the calcium ionophore 4BrA23187 was preceded by a rise of [Ca++]i to 1 mM in the PV. The [Ca++]i rise and the liberation of parasites from their host appear to be correlated. On the basis of these observations, we suggest that the increase of [Ca++]i in the vacuole may act as a signal that triggers the egress of T. gondii.

This study documents a difference between cystic fibrosis human (CF-HTG) and normal human (HTG) tracheal gland cells: the ability of histamine to induce an increase of intracellular free calcium concentration [Ca2+]i was abnormally reduced in CF-HTG cells. The magnitude of the [Ca2+]i peak rise in response to histamine is smaller in CF-HTG cells than in HTG cells, and the percentage of CF-HTG cells that increase [Ca2+]i is decreased compared with HTG cells. In contrast to histamine, the human neutrophil elastase (HNE) stimulation of both CF-HTG and HTG cells generated [Ca2+]i asynchronous oscillations and the magnitude of the peak [Ca2+]i response as well as the percentage of responding cells were similar for both groups. By videomicroscopy observations, the secretory response (exocytosis of secretion granules) of CF-HTG cells occurred with HNE, but not with histamine, thus suggesting that [Ca2+]i asynchronous oscillations may be linked to the exocytosis process in human tracheal gland cells.

Confocal microspectrofluorometry allows the analysis of fluorescent molecules such as anthracylines in isolated living cells. An optical microscope fitted with a phase-contrast 100 X water-immersion objective enables simultaneous observation of the sample, focusing of the laser beam on the selected cell fraction (nucleus) and collection of the fluorescence emitted from the sample. The resulting intranuclear spectra are interpreted according to a quantitative model of the fluorescence spectra of both free and DNA-bound anthracycline. The intranuclear drug concentration can thus be determined. This technique has been applied to blast cells collected in patients with acute leukemia. Leukemic cells are aspirated from bone marrow, separated by Ficoll sedimentation and resuspended in RPMI-1640 containing 10% fetal calf serum and 200 nM tetrahydropyranyl-doxorubicin (THP-DOX). After one hour, 20 cells are analyzed and the mean nuclear drug content is determined (C1). Cells are then resuspended in the same medium but without anthracycline for 3 hours and the mean intranuclear drug concentration is then also determined (C3). From C1 and C3 the retention rate (RR) is calculated. Firstly, the accuracy of the method was checked. In 4 AML patients, two different samples aspirated on the same day were divided into two portions. Thus, two measurements were made on each one (4 values per patient). Coefficients of variation were satisfactory (4, 6, 12, and 12%). Secondly, blast cells collected in patients with AML and ALL at diagnosis or in relapse were studied. P-glycoprotein (P-gp) and CD34 expression was also studied using respectively immunohistochemistry land flow cytometry. Results obtained from the first 21 patients showed that there was no correlation between RR and either P-gp or CD34 expression. This could result from the efflux of THP-DOX by other mechanisms and/or low sensitivity of the staining technique.

Microspectrofluorometry allows the analysis of fluorescent molecules such as anthracyclines in the nucleus of isolated living cells. Using this technique, we confirmed that the amount of doxorubicin or THP-doxorubicin incorporated into the nucleus was related to the resistant or sensitive character of K562 cells. It was then extended to the study of fresh leukemic cells and kinetic studies were performed allowing the calculation of the retention rate (RR) of anthracycline (THP-doxorubicin) into the cell nucleus. A reproducibility study confirmed the accuracy of the method. Blast cells collected in patients with acute myeloid (n = 22) or lymphoid (n = 8) leukemia, at diagnosis (n = 26), or in relapse (n = 4) have been studied. RR varied from 8 to 98% independently of the type of leukemia or the clinical status. RR did not correlate either with P-glycoprotein or with CD34 expression although this latter result should be confirmed on a higher number of subjects. Among 18 patients presenting with AML at diagnosis, 14 have been treated with intensive chemotherapy including anthracyclines; the only one who had resistant disease had the lowest RR value. In conclusion, the results obtained here show that microspectrofluorometry allows the performance of kinetic studies on fresh leukemic cells in order to quantify chemo-resistance phenomena related to drug transport.

Measurements of the intracellular free calcium concentration [Ca2+]i in single cells of the human tracheal gland cell line MM 39 demonstrate dynamic changes in [Ca2+]i after their exposure to human neutrophil elastase (HNE). A heterogeneity in [Ca2+]i responses measured cell to cell in monolayer culture is evident: cells generate an initial [Ca2+]i peak rise with or without a delayed time (up to 180 sec) followed either by a rapid return to baseline, asynchronous oscillations or a sustained plateau phase. From basal concentration of 85 +/- 15 nM, HNE (1 microM) produces a [Ca2+]i increase of 91 +/- 66 nM in about 50% of responding cells. At lower concentrations of HNE (0.1 microM, 0.01 microM), the [Ca2+]i rise remains similar, but only 30-40% of the cells are responding. Pretreatment of cells with the recombinant elafin protein, a specific elastase inhibitor, reduces both the [Ca2+]i response to HNE and the number of responding cells. Electron microscopy observations reveal an increased number of secretory granules located beneath the cell plasma membrane after HNE treatment. These results suggest that intracellular [Ca2+]i changes may be associated to the HNE-induced exocytosis in human tracheal gland cells. These findings could have implications with regard to the pathogenesis of increased mucus secretion in human airway diseases.

Confocal UV-microspectrofluorometry has been applied to measure fluorescence emission spectra of Indo-1 for the intracellular determination of free calcium ([Ca2+]i). To perform in situ calibrations of [Ca2+]i in the nucleus and the cytoplasm, Indo-1 has been loaded into living cells under its esterified form Indo-1/AM. For each controlled [Ca2+]i, intranuclear and intracytoplasmic spectra show a systematic blue shift, as compared with spectra in solution at the same [Ca2+]. In the Ca2+ saturated condition, the intranuclear spectra are more blue-shifted than in the cytoplasm. Thus, distinct in situ calibration curves have been distinguished for the nucleus and the cytoplasm. To calculate [Ca2+]i, intracellular spectra of Indo-1 have been characterized by two distinct methods. First, the ratio of emission intensities at 410 and 500 nm has been determined. Secondly, the analyzed spectrum has been decomposed into a linear combination of in situ free and Ca-bound Indo-1 reference spectra from the considered cellular compartment. Satisfactory spectral decompositions have been observed for each [Ca2+]i. The subcellular calibration curves allow one to determine the product of both intracellular constants, i.e. the ratio of quantum yields between free and Ca-bound Indo-1 and the apparent dissociation constant for Ca2+. The product of these constants has been shown to be similar in both subcellular compartments and in a buffered solution. The two methods used for the [Ca2+]i determination lead to equivalent results on unpermeabilized KB cells. They show a 1.3 times higher [Ca2+]i in the cytoplasm than in the nucleus (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Pre-resonance Raman spectroscopy has been applied to compare the vibrational modes of the R and S chiral isomers of 1-deaza-7,8-dihydropteridine when they are bound to tubulin. The main Raman bands are due to the chromophore and are coupled with the pi-pi electronic transition of C = C and C = N vibrational stretching. On binding to tubulin, the Raman spectra of both isomers are modified. However, the modifications induced are different for each isomer. The Raman bands due to C = C stretching from the phenyl ring are more strongly modified for the bound R isomer than for the S isomer. This leads us to suggest that R and S isomers differ in terms of their orientation in front of the binding locus of tubulin. In fact, with respect to the orientation of the bulky methyl group, the chromophore of the R isomer is more likely to be positioned against the external surface of either tubulin or GTPase proteins, while that of the S isomer is likely to be positioned away from the surface. The conformational changes induced in tubulin by R and S isomers have also been studied by Fourier transform infrared spectroscopy and by the analysis of amide I and II absorption bands. Both enantiomers induce similar minor changes to the tubulin secondary structure, corresponding to a decrease in the disordered alpha-helical content and accompanied by an increase in the undefined conformation content.

A phenotype of resistance to the new vinca alkaloid Navelbine was induced in the J82 human bladder carcinoma cells. The resistance factor of the resistant cell line (J82-NVB) to Navelbine was 17. The resistance phenotype of these cells is not a multidrug-resistance (MDR) phenotype. J82-NVB cells lack overexpression of P-glycoprotein and cross-resistance to MDR drugs like doxorubicin, epipodophyllotoxins or colchicine. Navelbine efflux was similar in sensitive and resistant cells, and resistance could not be explained by a difference of drug accumulation in these two cell lines. The cells were cross-resistant to vinca alkaloids and taxoids whose targets are microtubules. Immunofluorescence study of microtubules showed that depolymerization occured for the same Navelbine concentration in sensitive and resistant cells. This concentration induced growth inhibition in sensitive but not in resistant cells. Moreover, depolymerization induced by Navelbine treatment was reversible, after drug removal, in resistant cells only. This study suggests that J82-NVB cell resistance mechanism involves alterations of microtubule dynamics, allowing recovery of microtubules functions after treatment.

Scanning microspectrofluorometry has been developed to perform the mapping of fluorescence spectra from all locations in a living cell. This new method has been applied to study the molecular environment of rhodamine 123 (R123) in sensitive (K562, CEM) and multidrug-resistant (K562-R, CEM/VLB100) tumor cells. All cells exposed to R123 showed a similar distribution of fluorescence in the perinuclear region. A lower cytoplasmic fluorescence intensity corresponding to a reduced drug accumulation was observed in resistant cells, as expected in the multidrug resistance process. Fluorescence emission spectra of R123 are useful to probe the polarity of the R123 environment. Thus, fluorescence spectra of R123-treated cells have been analyzed as a linear combination of model spectra: R123 in water and R123 in tensio-active Triton X-100. In sensitive cells, emission spectra of R123 underwent a red shift, equivalent to those observed in isolated coupled mitochondria. This suggests the formation of a complex in hydrophobic sites. In contrast, R123 spectra were less shifted in resistant cells, showing two types of both hydrophobic and hydrophilic binding sites. This could be related to an intracellular redistribution of R123 in resistant cells.

The protonation state and intracellular distribution of ellipticine were investigated in single human mammary T47D cells by confocal laser microspectrofluorimetry. In the cell nucleus, only the protonated form of ellipticine was detected as a direct consequence of its apparent pK increase upon DNA binding. Both protonated and neutral forms were present in the aqueous cytoplasm, where the pH is close to the drug pK. When cells were incubated in high concentrations of K+, a condition that depolarizes the plasma membrane potential, ellipticine cellular accumulation was reduced. In the cytoplasm, ellipticine was mainly bound to mitochondria, and its protonation equilibrium was shifted toward the neutral form. The fluorescence spectrum of ellipticine bound to mitochondria was insensitive to valinomycin, whereas it was markedly shifted toward the protonated form after carbonyl cyanide p-trifluoromethoxy-phenylhydrazone or nigericin addition. Similar studies with ellipticine bound to isolated mitochondria suggest that it behaves as a fluorescent probe of mitochondrial pH in both isolated mitochondria and single living cells.

A high-performance liquid chromatographic method has been developed for the determination within tumour cells of a new vinca alkaloid, vinorelbine. Extractions of vinorelbine from cells were carried out using absolute ethanol. The extracts were injected into a reversed-phase system consisting of two Novapak C18 columns connected in series. The mobile phase was acetonitrile-phosphate buffer, pH 2.7 (60:40, v/v). Using a fluorescence detection, the limit of determination was 8 pmol injected. This method would be suitable for studying the cellular pharmacokinetics of vinorelbine in patients.

We have demonstrated that in vitro resistance of tumor cells to doxorubicin (Dox) can be fully circumvented by using doxorubicin-loaded nanospheres (Dox-NS), consisting of biodegradable polyisohexylcyanoacrylate polymers of 300 nm diameter and containing 2.83 mg of Dox per 31.5 mg of polymer. Five different multidrug-resistant cell lines, characterized by mdr1 amplification, were used in this study: Dox-R-MCF7, a human breast adenocarcinoma; SKVBL1, a human ovarian adenocarcinoma; K562-R, a human erythroleukemia; and two murine lines: P388-Adr-R, a monocytic leukemia of DBA2 mouse, and LR73MDR, a Chinese hamster ovarian cell line. These lines were 38.7, 210, 232, 143 and 20 times more resistant than their corresponding sensitive counterparts, respectively. Using Dox-NS, we obtained complete reversion of drug resistance in vitro, i.e. cell growth inhibition comparable with that obtained with sensitive cells exposed to free Dox. In vivo, we significantly prolonged the survival of DBA2 mice which had previously received P388-Adr-R cells by i.p. injections of Dox-NS, while free Dox injection was ineffective toward this rapidly growing tumor. (Prolongation of survival time: 115% vs 167% after Dox vs Dox-NS treatment, respectively.) Using the MCF7 cell line and its resistant variant, we studied the intracellular concentration and the cytoplasmic and nuclear distribution of Dox by laser microspectrofluorometry (LMSF). In sensitive cells, we observed a similar accumulation and distribution of Dox whatever the form of Dox delivery, i.e. whether free or carried by nanospheres. Analysis by LMSF showed that 99% of intranuclear Dox was bound to DNA after treatment with both forms of Dox. Of Dox, 81 and 83% were found in the intranuclear compartment of sensitive cells incubated with free Dox and Dox-NS, respectively. Resistant cells incubated with Dox-NS accumulated the same amount of Dox as sensitive cells incubated with free Dox or with Dox-NS. Dox, when loaded in nanospheres, bypasses the efflux mechanism responsible for multidrug resistance. LMSF analysis showed that Dox, transported and released by nanospheres, interacts with DNA identically in sensitive and resistant cells.

The accumulation of doxorubicin (DOX) in white blood cells of treated patients has been studied by quantitative microspectrofluorometry. From blood samples of treated patients, leucocyte subpopulations were separated by the gradient method. Emission fluorescence spectra from a microvolume of a single living cell nucleus were analysed in terms of spectral shape and fluorescence yield between free and DNA-bound doxorubicin. With this non-destructive analysis technique, intranuclear doxorubicin concentrations were determined within +/- 10%. Doxorubicin concentrations were measured in patients treated with bolus injection. After an accumulation of DOX in leucocytes during the first 30 min, intranuclear doxorubicin concentration did not vary significantly for 24 h, whereas its concentration in plasma decreased. Despite large differences between patients, monocytes accumulated significantly more doxorubicin than granulocytes or lymphocytes did.

Two colon cancer cell lines, HT-29 (human) and DHD/K12/TRb (rat), were grown as monolayer cultures to various confluence degrees. The cytotoxic efficacies of doxorubicin and 4'-deoxydoxorubicin, evaluated by a survival assay, and the nuclear drug concentrations, measured by microspectrofluorometry, were shown to progressively decrease with the augmentation of confluence. This confluence dependent resistance (CDR) to anthracyclines was demonstrated independent of the multidrug resistance drug efflux mechanism. The cellular uptake of three compounds (sodium [51Cr]chromate, D-[14C]alanine, L-[14C]glucose) known to passively diffuse across the cell membrane as anthracyclines do was also reduced in confluent cells. After trypsin cell detachment, the kinetics of reversion of the sodium [51Cr]chromate uptake decrease and that of CDR were similar. Therefore, CDR may be attributed to a reduction of anthracycline cell intake due to a general alteration of passive diffusion across the cell membrane. However, CDR is only partly explained by this phenomenon since a reduced sensitivity of confluent cells was observed compared with nonconfluent cells for a similar amount of drug in their nuclei. CDR could explain the high resistance to anthracyclines of some solid tumors, such as colon tumors, in which cancer cells are tightly aggregated.