Analysis of the distribution of MRI contrast agents in the livers of small animals by means of complementary microscopies.

BACKGROUND: Magnetic resonance imaging (MRI) contrast agents contain magnetic molecules such as iron (Fe) or gadolinium (Gd) that are injected in vivo into rats or mice to study their distribution inside the liver. Fluorescent europium (Eu) can be used as a model of Gd to obtain comparable information of this distribution of corresponding contrast agents. In a similar approach, Fe can be attached to Texas Red and used as a model of ferumoxides and be detected by fluorescence. METHODS: To combine and compare the advantages of different microscopic imaging modes, characterization studies were carried out by means of a confocal laser scanning microscope (CLSM), a secondary ion mass spectrometric (SIMS) microscope, and an electron energy loss spectrometric (EELS) microscope. In the case of CLSM, the locations of fluorescent signals inside preparations were determined by factor analysis of biomedical image sequences (FAMIS) and selection of image sequences at emission. RESULTS: By CLSM and FAMIS, we distinguished chelated Eu and Texas Red attached to Fe. By SIMS microscopy, we distinguished Eu and Gd of chlorides and chelates and Fe of a ferumoxide. By EELS microscopy, we distinguished Eu and Gd of chlorides. CONCLUSIONS: Analysis of compounds inside correlative specimens by means of CLSM, SIMS, and EELS microscopes provided complementary results.

Human immunodeficiency virus type 1 central DNA flap: dynamic terminal product of plus-strand displacement dna synthesis catalyzed by reverse transcriptase assisted by nucleocapsid protein.

To terminate the reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome, a final step occurs within the center of the proviral DNA generating a 99-nucleotide DNA flap (6). This step, catalyzed by reverse transcriptase (RT), is defined as a discrete strand displacement (SD) synthesis between the first nucleotide after the central priming (cPPT) site and the final position of the central termination sequence (CTS) site. Using recombinant HIV-1 RT and a circular single-stranded DNA template harboring the cPPT-CTS sequence, we have developed an SD synthesis-directed in vitro termination assay. Elongation, strand displacement, and complete central flap behavior were analyzed using electrophoresis and electron microscopy approaches. Optimal conditions to obtain complete central flap, which ended at the CTS site, have been defined in using nucleocapsid protein (NCp), the main accessory protein of the reverse transcription complex. A full-length HIV-1 central DNA flap was then carried out in vitro. Its synthesis appears faster in the presence of the HIV-1 NCp or the T4-encoded SSB protein (gp32). Finally, a high frequency of strand transfer was shown during the SD synthesis along the cPPT-CTS site with RT alone. This reveals a local and efficient 3'-5' branch migration which emphasizes some important structural fluctuations within the flap. These fluctuations may be stabilized by the NCp chaperone activity. The biological implications of the RT-directed NCp-assisted flap synthesis are discussed within the context of reverse transcription complexes, assembly of the preintegration complexes, and nuclear import of the HIV-1 proviral DNA to the nucleus toward their chromatin targets.

Determinants of Mg2+-dependent activities of recombinant human immunodeficiency virus type 1 integrase.

The relationship between Mg(2+)-dependent activity and the self-assembly state of HIV-1 integrase was investigated using different protein preparations. The first preparations, IN(CHAPS) and IN(dial), were purified in the presence of detergent, but in the case of IN(dial), the detergent was removed during a final dialysis. The third preparation, IN(zn), was purified without any detergent. The three preparations displayed comparable Mn(2+)-dependent activities. In contrast, the Mg(2+)-dependent activity that reflects a more realistic view of the physiological activity strongly depended on the preparation. IN(CHAPS) was not capable of using Mg(2+) as a cofactor, whereas IN(zn) was highly active under the same conditions. In the accompanying paper [Deprez, E., et al. (2000) Biochemistry 39, 9275-9284], we used time-resolved fluorescence anisotropy to demonstrate that IN(CHAPS) was monomeric at the concentration of enzymatic assays. Here, we show that IN(zn) was homogeneously tetrameric under similar conditions. Moreover, IN(dial) that exhibited an intermediary Mg(2+)-dependent activity existed in a monomer-multimer equilibrium. The level of Mg(2+)- but not Mn(2+)-dependent activity of IN(dial) was altered by addition of detergent which plays a detrimental role in the maintenance of the oligomeric organization. Our results indicate that the ability of integrase to use Mg(2+) as a cofactor is related to its self-assembly state in solution, whereas Mn(2+)-dependent activity is not. Finally, the oligomeric IN(zn) was capable of binding efficiently to DNA regardless of the cationic cofactor, whereas the monomeric IN(CHAPS) strictly required Mn(2+). Thus, we propose that a specific conformation of integrase is a prerequisite for its binding to DNA in the presence of Mg(2+).

Efficient DNA transfection mediated by the C-terminal domain of human immunodeficiency virus type 1 viral protein R.

Viral protein R (Vpr) of human immunodeficiency virus type 1 is produced late in the virus life cycle and is assembled into the virion through binding to the Gag protein. It is known to play a significant role early in the viral life cycle by facilitating the nuclear import of the preintegration complex in nondividing cells. Vpr is also able to interact with nucleic acids, and we show here that it induces condensation of plasmid DNA. We have explored the possibility of using these properties in DNA transfection experiments. We report that the C-terminal half of the protein (Vpr(52-96)) mediates DNA transfection in a variety of human and nonhuman cell lines with efficiencies comparable to those of the best-known transfection agents. Compared with polylysine, a standard polycationic transfection reagent, Vpr(52-96) was 10- to 1,000-fold more active. Vpr(52-96)-DNA complexes were able to reach the cell nucleus through a pH-independent mechanism. These observations possibly identify an alternate pathway for DNA transfection.

Properties and growth mechanism of the ordered aggregation of a model RNA by the HIV-1 nucleocapsid protein: an electron microscopy investigation.

NCp7, the nucleocapsid protein of the human immunodeficiency virus type 1, induces an ordered aggregation of RNAs, a mechanism that is thought to be involved in the NCp7-induced promotion of nucleic acid annealing. To further investigate this aggregation the morphology and the properties of the NCp7-induced aggregates of the model RNA homoribopolymer, polyA, were investigated by electron microscopy in various conditions. In almost all the tested conditions, the aggregates were spherical and consisted of a central dense core surrounded by a less dense halo made of NCp7-covered polyA molecules. The formation of these aggregates with a narrow distribution of sizes constitutes a distinctive feature of NCp7 over other single-stranded nucleic acid binding proteins. In most conditions, at the shortest times that can be reached experimentally, all the polyA molecules were already incorporated in small aggregates, suggesting that the nucleation step and the first aggregation events took place rapidly. The aggregates then orderly grew with time by fusion of the smaller aggregates to give larger ones. The aggregate halo was important in the fusion process by initiating the bridging between the colliding aggregates. In the presence of an excess of protein, the aggregates grew rapidly but were loosely packed and dissociated easily, suggesting adverse protein-protein interactions in the aggregates obtained in these conditions. In the presence of an excess of nucleotides, the presence of both amorphous nonspherical and slowly growing spherical aggregates suggested some changes in the mechanism of aggregate growth due to an incomplete covering of polyA molecules by NCp7. Finally, we showed that in the absence of added salt, the aggregate fusions were unfavored but not the initial events giving the first aggregates, the reverse being true in the presence of high salt concentrations (> or = 300 mM).

Intracellular signaling events in CD77-mediated apoptosis of Burkitt's lymphoma cells.

In the hematopoietic system CD77, a glycolipid surface antigen, is restricted to group I Burkitt's lymphoma (BL) cell lines and a subset of germinal center B lymphocytes. Recently, we have reported that recombinant B subunits of Verotoxin, which specifically binds to CD77, induce programmed cell death of CD77+ BL cells. Here, we show that an anti-CD77 monoclonal antibody (38.13) immobilized on tissue culture dishes also induces apoptosis, and we have explored the signal transducing events leading to this cell death. We show that ligation of CD77 antigen causes an increase of the intracellular Ca2+ concentration owing to an influx of extracellular Ca2+ through calcium channels. Chelation of extracellular Ca2+ with EGTA partially prevents anti-CD77-induced apoptosis, indicating that this process is probably Ca2+ dependent. We show that the cross-linking of CD77 provokes an increase of intracellular cAMP levels followed by cAMP-dependent protein kinase activation. We report that BL cells produce ceramide when they are exposed to 38.13 but, unexpectedly, without a concomitant decrease in sphingomyelin or CD77 content. Finally, we provide evidence that C2-ceramide, calcium ionophore, and forskolin (which increases intracellular levels of cAMP) independently induce apoptosis of CD77+ BL cells and, moreover, that C2-ceramide and forskolin strongly synergize to cause cell death. The possible role of CD77-mediated apoptosis in the B cell selection that occurs in germinal centers is discussed.

Effect of retinoic acid on HL-60 cells infected with human immunodeficiency virus type 1.

HL-60 cells infected with human immunodeficiency virus type 1 (HIV 1) can be induced to differentiate along the granulocyte pathway by retinoic acid. In these cells, HIV mRNA synthesis is stimulated, but synthesis of viral proteins and virus replication are blocked and HIV-infected cells die after becoming apoptotic and/or vacuolized.

Electron microscopy of the nucleocapsid from disrupted Moloney murine leukemia virus and of associated type VI collagen-like filaments.

To analyze the constituents of retroviruses, the Moloney murine leukemia virus was disrupted and observed by dark-field electron microscopy. Virus disruption was achieved by several methods: osmotic shock, freezing-thawing cycles, and exposure to urea up to 4 M, to NaCl up to 1 M, and to Triton X-100. Several components associated with broken Moloney murine leukemia virus were repeatedly found in preparations. These components have been described as rings, thick filaments, chain-like filaments, threads covered with proteins, threads with buckles, and naked threads. A quantitative analysis of the occurrence of these components has been carried out. Among them, the thick filaments composed of a compact helical arrangement of small beads 5 nm in diameter were considered to represent the nucleocapsid. The protease-sensitive buckles found on some threads could be a compact form of the viral RNA associated to the nucleocapsid protein NCp10. The RNase-sensitive naked threads are interpreted as the deproteinized viral RNA itself. The ubiquitous chain-like filaments possess a periodic structure identical to that of polymerized type VI collagen. It is proposed that this adhesive protein is associated with the viral envelope taken from the cell membrane during the budding process of retroviruses.

Replication of human immunodeficiency virus in HL-60 cells.

HL-60 cells carrying the CD4 marker could be productively infected with human immunodeficiency virus (HIV) but did not form syncytia, though 11-14 days p.i., there was a transient decrease in cell multiplication and viability. After prolonged passage, a subpopulation of HL-60 cells was selected. The virus produced differed from the initial input virus grown on CEM cells: the virions lacked knobs, were either empty or had abnormal cores, had a higher ratio p24/gp41,gp110 and were less infectious. After prolonged passage, virus was produced which was fully infectious for CEM but not for fresh HL-60 cells, and which ressembled the input virus with respect to morphology and p24/gp41,gp110 ratio.

Electron microscopic observations of the effects of anthraquinone derivatives on plasmid DNA.

Electron microscopy was used to analyse the precipitation of DNA observed when mixed with two tripeptide derivatives of mitoxantrone, with or without a 5,8-dihydroxy group (DHQ-GHK and Q-GHK, respectively) on the anthraquinonic ring. This precipitation was compared to that obtained with the basic drugs, mitoxantrone (DHAQ) and ametantrone (AQ). The effects of these compounds on the supercoiling of form I and the lengthening of form II of pBR322 DNA molecules, respectively, were evaluated. A strong lengthening of the DNA molecules was observed for ametantrone (max: 57%), but only 32% for Q-GHK, both at r (drug/base pari) = 250. With the dihydroxy derivative DHQ-GHK, it was not possible to show more than a 10% increase in length because DNA molecules were not measurable at r greater than 100. Only Q-GHK relaxed supercoiled molecules at the low r values of 10. Complex phenomena of condensation-precipitation were observed with these two tripeptide derivatives. In addition to a strong lengthening of form II DNA molecules, AQ induced specifically the formation of toruses, and DHAQ that of large organized DNA condensation. The variety of the aggregations is described and discussed with regard to the antitumor properties of these derivatives, and the literature concerning the various descriptions of DNA aggregation.

Inhibition of human immunodeficiency virus (HIV) type 1 multiplication by an avian cellular factor.

A factor secreted from avian cells infected non productively with a non cytopathogenic mutant of vesicular stomatitis virus (VSV ts 1026) interferes with HIV replication in CEM cells and peripheral blood monocytes (PBL). Production of infectious particles is decreased and many virions lack cores and/or spikes. In CEM cells the prmRNA is spliced into 7.5, 4, and 2 kb mRNA. Residual virus contains less env encoded proteins and p 18; p 25 appears as several bands. The processing of tat, rev. and nef proteins differs in treated cells and in controls.

CD77: an antigen of germinal center B cells entering apoptosis.

We have previously reported that a neutral glycolipid (globotriosylceramide; Gb3) was specifically expressed on Burkitt's lymphoma cells and on a subset of germinal center tonsillar B lymphocytes. Recently the Gb3 molecule was recognized as a new B cell differentiation antigen and now defines the CD77 cluster. Here we report an extensive phenotypic and functional characterization of the tonsillar CD77+ B lymphocytes. These cells have a low buoyant density and are thus purified using a Percoll gradient. They express various B cell antigens such as CD19, CD20, CD21, CD22 and CD40, as well as the adhesion molecules LFA-1, LFA-3 and CD44. They are positive for surface IgM and negative for surface IgD. Although these results suggest a phenotype of activated B cells, the CD77+ cells are negative for the classical activation antigens: CD23 (the low-affinity Fc receptor for IgE), CD25 [the interleukin (IL) 2 receptor alpha chain] and CD71 (the transferrin receptor). Proliferation and protein synthesis of CD77+ cells was measured after stimulation with a range of mitogens and IL. None of the agents tested are able to induce proliferation and protein synthesis with the exception of a combination of recombinant IL 4 plus anti-CD40 antibody. When examined by electron microscopy, CD77+ B lymphocytes present a morphology similar to that of cells undergoing programmed cell death, also called apoptosis (i.e. chromatin condensation, nuclear fragmentation, membrane blebbing). As shown by direct examination of DNA, these CD77+ cells are indeed in the process of apoptosis. Treatment of the CD77+ cells by recombinant IL 4 and anti-CD40 antibody prevents apoptosis. All these results suggest that the CD77 molecule defines a B lymphocyte maturation pathway, specific for germinal center, where the cells undergo programmed cell death.

SV40 induced cellular immortalization: phenotypic changes associated with the loss of proliferative capacity in a conditionally immortalized cell line.

Immortalization of rodent embryo fibroblasts by SV40 is dominantly maintained by the large T antigen. The aim of this work is to characterize some of the events associated with the loss of proliferative capacity in a rat cell line, called REtsAF, which is conditionally immortalized by the tsA58 allele of SV40 large T antigen. DNA replication is arrested less than 24 h after the shift to the restrictive temperature (39 degrees C). This arrest occurs without specificity relative to the cell cycle stage, which suggests that a function essential throughout the cell cycle is affected. A two-dimensional SDS polyacrylamide gel electrophoresis analysis of proteins shows that, although the global rate of protein synthesis is only slightly affected at 39 degrees C, the rate of accumulation of specific proteins is either increased or decreased. Finally we present biochemical and electron microscopy data showing that alterations of the mitochondria occur upon shift to 39 degrees C.

Location of B- and Z-DNA in the chromosomes of a primitive eukaryote dinoflagellate.

The usual conformation of DNA is a right-handed double helix (B-DNA). DNA with stretches of alternating purine-pyrimidine (G-C or A-T) can form a left-handed helix (Z-DNA). The transition B----Z, facilitated by the presence of divalent cations, cytosine methylation, or constraints on DNA such as superhelicity may play a role in the regulation of gene expression and/or in DNA compaction (Zarling, D. A., D. J. Arndt-Jovin, M. Robert-Nicoud, L. P. McIntosh, R. Tomae, and T. M. Jovin. 1984. J. Mol. Biol. 176:369-415). Divalent cations are also important in the structure of the quasi-permanently condensed chromosomes of dinoflagellate protists (Herzog, M., and M.-O. Soyer. 1983. Eur. J. Cell Biol. 30:33-41) which also have superhelicity in their DNA. The absence of histones in dinoflagellate chromosomes suggest that the search for Z-DNA sequences might be fruitful and could provide one indication of the physiological role of this particular DNA conformation. We report a complete immunofluorescent and immunogold analysis of the nuclei of the dinoflagellate Prorocentrum micans E. using monoclonal and polyclonal anti-B and anti-Z-DNA antibodies. Positive labeling was obtained with immunofluorescence using squash preparations and cryosections, both of which showed the intranuclear presence of the two DNA conformations. In ultrathin sections of aldehyde-prefixed, osmium-fixed, and epoxy-embedded cells, we have localized B-DNA and Z-DNA either with single or double immunolabeling using IgG labeled with 5- and 7-nm gold particles, respectively. Chromosomal nucleofilaments of dividing or nondividing chromosomes, as seen in ultrathin sections in their arch-shaped configuration, are abundantly labeled with anti-B-DNA antibody. Extrachromosomal anti-B-DNA labeling is also detected on the nucleoplasm that corresponds to DNA loops; we confirm the presence of these loops previously described external to the chromosomes (Soyer, M.-O., and O. K. Haapala. 1974. Chromosoma (Berl.). 47:179-192). B labeling is also visible in the nucleolus organizer region (NOR) and in the fibrillo-granular area (containing transcribing rDNA) of the nucleolus. Z-DNA was localized in limited areas inside the chromosomes, often at the periphery and near the segregation fork of dividing chromosomes. In the nucleolus, Z-DNA is observed only in the NOR area and never in the fibrillo-granular area. For both types of antibody experiments, controls using gold-labeled IgG without primary antibody were negative. A quantitative evaluation of the distribution of the gold-labeled IgG and a parametric test support the validity of these experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

Selective alteration of mitochondrial function by Ditercalinium (NSC 335153), a DNA bisintercalating agent.

The bifunctional intercalator Ditercalinium (NSC 335153) demonstrates an anti-tumoral cytotoxicity markedly different from other intercalating agents. A delayed toxicity is observed in eucaryotic cells, both in vitro and in vivo, at drug concentrations far below those required to observe immediate toxic effects. Fluorescence microscopy demonstrates that Ditercalinium and the mitochondrial-staining fluorophore DiOC2(5) are concentrated in the same cellular organelles of L1210 cells. Electron microscopy of Ditercalinium-treated cells reveals extensive and progressive swelling of mitochondria, with no other ultrastructural changes observed. Ditercalinium uptake and toxicity are in part related to mitochondrial membrane potential. However, drug accumulation itself does not immediately alter the mitochondrial membrane potential. Cellular ATP pool levels and the rate of respiration fall progressively after drug treatment. Nucleotide pools in DC3F cells, measured between drug treatment and death, show marked drops in pyrimidine levels while purine nucleotide levels decline more slowly. Addition of uridine or cytidine partially rescues Ditercalinium-treated cells, while toxicity is increased in the presence of 2-deoxyglucose. The combined evidence indicates that the toxicity of Ditercalinium to murine leukemia cells (L1210) and Chinese Hamster lung cells (DC3F) is due to disruption of mitochondrial function.

Cytoplasmic accumulation of ditercalinium in rat hepatocytes and induction of mitochondrial damage.

Ditercalinium (NSC 335153), a 7H-pyridocarbazole dimer, is a bis-intercalating agent whose mechanism of action differs from that of other mono-intercalating compounds, such as ellipticine derivatives. After a Phase I clinical trial, where irreversible hepatotoxicity was the dose-limiting side-effect, we have reinvestigated the disposition of ditercalinium in rats after i.v. administration. Tissue distribution of the tritiumlabeled drug was studied during 5 weeks. The drug distributed very quickly into tissues, and accumulated mostly in liver and kidneys. A much slower clearance followed, with a half-life greater than 7 days. The present study raises the question of whether ditercalinium, a biscationic lipophilic agent, exerts a direct mitochondria interaction both in vitro and in vivo. Fluorescence microscopy on rat tissue cryosections, after drug administration, showed that the major cellular site of drug accumulation corresponds to mitochondria. The mitochondrial probe 3,3'-diethyloxadicarbocyanine confirmed the mitochondrial localization of ditercalinium. An identical fluorescent pattern was found in cultured rat hepatocytes. These fluorescent granulation patterns suggest mitochondrial damage. To further study cell alterations, ultrastructural changes in rat liver and kidneys were observed with selective mitochondrial damage while nuclei remained apparently normal. The same observations were made in rat hepatocyte cultures. Therefore, the accumulation of ditercalinium in tissues and, more particularly, in mitochondrial probably plays an important role in ditercalinium-induced toxicity.

Selective loss of mitochondrial DNA after treatment of cells with ditercalinium (NSC 335153), an antitumor bis-intercalating agent.

Ditercalinium (NSC 335153), a bifunctional intercalating molecule with antitumor activity, is found to express its toxicity through a mechanism of action completely different from that of other monointercalating agents. Electron microscopic observation of ditercalinium-treated cells shows a drastic alteration of mitochondrial structure. Cells deficient in mitochondrial respiration (GSK3 cells) isolated by A. Franchi et al. (Int. J. Cancer, 27: 819-827, 1981) are about 25-fold more resistant than cells deficient in glycolysis (DS7 cells) isolated by J. Pouysségur et al. (Proc. Natl. Acad. Sci. USA, 77: 2698-2701, 1980). Revertants have been isolated from GSK3 cells. In these cells, the sensitivity to ditercalinium has been recovered with mitochondrial respiration. Ditercalinium treatment of L1210 leukemic mouse cells leads to a specific elimination of mitochondrial DNA detected by DNA-DNA hybridization. No measurable alteration of nuclear DNA is observed. In contrast, the monomeric analogue of ditercalinium only alters nuclear DNA and does not change the mitochondrial DNA content. The activity of cytochrome c oxidase, an enzyme which contains a subunit coded by the mitochondrial DNA, decreases exponentially in treated cells with a half-life of 24 h, corresponding to the turnover of the enzyme. These results suggest that ditercalinium exerts a specific cytotoxic effect at the level of mitochondrial DNA. This action could account for the delayed cytotoxicity induced by this compound.

Electron microscopy of the reactions of anti-poly A. poly U and anti-poly I. poly C antibodies with synthetic polynucleotide complexes and natural nucleic acids.

The reactions between purified anti-poly A. poly U and-poly I. poly C. antibodies (IgG and IgM), and synthetic and natural polynucleotides were visualized at the molecular level. This was achieved by the use of fine tungsten bidirectional shadowing of molecules adsorbed onto thin carbon films, combined with dark field electron microscopic observation. A progression was observed from monogamous multivalency (binding of a single multifunctional antigen molecule with several combining sites of the same antibody molecule simultaneously) (Crothers and Metzger, 1972, Immunochemistry, 9, 341-357), to aggregation. Different types of figures were observed, among which loops formed by the coiling of the antigen around a single IgM molecule were very frequently seen. The tendency of IgG antibodies to bind cooperatively to certain antigens was also noted. In contrast, cross-links were seldom encountered. The cross-reactivity of different polynucleotides was also assessed by a quantitative analysis. The length of antigen associated to an antibody molecule (either IgG or IgM) was also measured.