Optical probing of sodium dynamics in neurons and astrocytes.

Changes in intracellular Na(+) concentration underlie essential neurobiological processes, but few reliable tools exist for their measurement. Here we characterize a new synthetic Na(+)-sensitive fluorescent dye, Asante Natrium Green (ANG), with unique properties. This indicator was excitable in the visible spectrum and by two-photon illumination, suffered little photobleaching and located to the cytosol were it remained for long durations without noticeable unwanted effects on basic cell properties. When used in brain tissue, ANG yielded a bright fluorescent signal during physiological Na(+) responses both in neurons and astrocytes. Synchronous electrophysiological and fluorometric recordings showed that ANG produced accurate Na(+) measurement in situ. This new Na(+) indicator opens innovative ways of probing neuronal circuits.

Avoiding epigenetic variations in transgene expression with genetic insulators

Gene transfer that relies on integrating vectors often suffers from epigenetic or regulatory effects that influence the expression of the therapeutic gene and=or of cellular genes located near the vector integration site in the chromosome. Insulator elements act to block gene activation by enhancers, while chromatin domain boundary or barrier sequences prevent gene-silencing effects. At present, the modes of action of insulator and barriers are poorly understood, and their use in the context of gene therapies remains to be documented. Using combinations of reporter genes coding for indicator fluorescent proteins, we constructed assay systems that allow the quantification of the insulator or of the barrier activities of genetic elements in individual cells. This presentation will illustrate how these assay systems were used to identify short DNA elements that can insulate nearby genes from activation by viral vector enhancer elements, and=or that can block the propagation of a silent chromatin structure that leads to gene silencing. We will show that small elements of the order of 100-400 nucleotides can be designed to achieve both insulator and boundary function, as needed for safer integrating viral vectors.

Intravitreal ranibizumab in the treatment of choroidal neovascularization associated with idiopathic central serous chorioretinopathy.

PURPOSE: We evaluate the functional and anatomic outcome after intravitreal ranibizumab treatment in patients with choroidal neovascularization (CNV) related to chronic central serous chorioretinopathy (CSC). METHODS: This is a small case series of 5 eyes with CNV associated with chronic CSC treated with intravitreal injection of 0.5 mg ranibizumab in the Jules Gonin University Eye Hospital from July 2007 to July 2009. Baseline and monthly follow-up visits included best-corrected visual acuity (BCVA), fundus examination, and optical coherence tomography (OCT). Fluorescein and indocyanine green angiography (ICG) were performed at baseline and repeated at least every 6 months. RESULTS: We studied 5 eyes of 4 patients with a mean age of 66 years. Mean follow-up was 21 months (SD 1.9). The mean number of intravitreal injections administered for each patient was 10 (SD 4.6). The mean initial BCVA was 0.23 (decimal equivalent) (logMAR 0.64, SD 0.13). At the last follow-up, mean BCVA was 0.44 decimal equivalent (logMAR 0.36, SD 0.31). Mean central macular thickness (CMT) measured with OCT was 330 microm (SD 43) at baseline and decreased at the final follow-up to 243 microm (SD 44 ). Persistent intraretinal or subretinal fluid on OCT and/or multifocal areas of increased choroidal permeability on ICG angiographies were present in all patients at the last follow-up visit. CONCLUSIONS: Intravitreal ranibizumab appeared to be an effective treatment of CNV related to chronic CSC. However, residual intraretinal or subretinal fluid and increased choroidal permeability persisted. Prospective controlled studies are warranted to evaluate the long-term safety and efficacy of intravitreal ranibizumab.

Mechanism of reduction of virus release and cell-cell fusion in persistent canine distemper virus infection.

Canine distemper virus (CDV), a mobillivirus related to measles virus causes a chronic progressive demyelinating disease, associated with persistence of the virus in the central nervous system (CNS). CNS persistence of morbilliviruses has been associated with cell-to-cell spread, thereby limiting immune detection. The mechanism of cell-to-cell spread remains uncertain. In the present study we studied viral spread comparing a cytolytic (non-persistent) and a persistent CDV strain in cell cultures. Cytolytic CDV spread in a compact concentric manner with extensive cell fusion and destruction of the monolayer. Persistent CDV exhibited a heterogeneous cell-to-cell pattern of spread without cell fusion and 100-fold reduction of infectious viral titers in supernatants as compared to the cytolytic strain. Ultrastructurally, low infectious titers correlated with limited budding of persistent CDV as compared to the cytolytic strain, which shed large numbers of viral particles. The pattern of heterogeneous cell-to-cell viral spread can be explained by low production of infectious viral particles in only few areas of the cell membrane. In this way persistent CDV only spreads to a small proportion of the cells surrounding an infected one. Our studies suggest that both cell-to-cell spread and limited production of infectious virus are related to reduced expression of fusogenic complexes in the cell membrane. Such complexes consist of a synergistic configuration of the attachment (H) and fusion (F) proteins on the cell surface. F und H proteins exhibited a marked degree of colocalization in cytolytic CDV infection but not in persistent CDV as seen by confocal laser microscopy. In addition, analysis of CDV F protein expression using vaccinia constructs of both strains revealed an additional large fraction of uncleaved fusion protein in the persistent strain. This suggests that the paucity of active fusion complexes is due to restricted intracellular processing of the viral fusion protein.

Cell morphology and intracellular ionic homeostasis explored with a multimodal approach combining epifluorescence and digital holographic microscopy.

The authors have developed a live-cell multimodality microscope combining epifluorescence with digital holographic microscopy; it has been implemented with a decoupling procedure allowing to separately measure from the quantitative phase important cell parameters including absolute volume, shape and integral intracellular refractive index. In combination with the numerous different specific fluorescent cellular probes, this multimodality microscopy can address important issues in cell biology. This is demonstrated by the study of intracellular calcium homeostasis associated with the change in cell volume, which play a critical role in the excitotoxicity-induced neuronal death.

Myosin, tubulin and laminin immunoreactivity in the ectoderm of the growing area opaca of the chick embryo

Distribution of myosin, tubulin and laminin immunoreactive cells in the area opaca of the young chick embryo (Stages 4-8 HH) was studied using immunofluorescence technique. For the three markers, the number of stained cells increased with the age of the blastoderm. Cells stained for tubulin and laminin, were distributed throughout the area opaca, showing no supracellular organization. On the contrary, the cells stained for myosin became organized in a ring surrounding the area pellucida. This pattern appeared at the stage 6. Such an heterogenous distribution of the markers suggests a functional diversification of the ectodermal cell monolayer forming at these early developmental stages the area opaca. This idea is also supported by the results of autoradiography for tritiated thymidin which showed that the edge cells did not synthetize DNA and consequently did not divide.

Immunolocalization of GLUTX1 in the testis and to specific brain areas and vasopressin-containing neurons.

GLUTX1 or GLUT8 is a newly characterized glucose transporter isoform that is expressed at high levels in the testis and brain and at lower levels in several other tissues. Its expression was mapped in the testis and brain by using specific antibodies. In the testis, immunoreactivity was expressed in differentiating spermatocytes of type 1 stage but undetectable in mature spermatozoa. In the brain, GLUTX1 distribution was selective and localized to a variety of structures, mainly archi- and paleocortex. It was found in hippocampal and dentate gyrus neurons as well as amygdala and primary olfactory cortex. In these neurons, its location was close to the plasma membrane of cell bodies and sometimes in proximal dendrites. High GLUTX1 levels were detected in the hypothalamus, supraoptic nucleus, median eminence, and the posterior pituitary. Neurons of these areas synthesize and secrete vasopressin and oxytocin. As shown by double immunofluorescence microscopy and immunogold labeling, GLUTX1 was expressed only in vasopressin neurons. By immunogold labeling of ultrathin cryosections microscopy, GLUTX1 was identified in dense core vesicles of synaptic nerve endings of the supraoptic nucleus and secretory granules of the vasopressin positive neurons. This localization suggests an involvement of GLUTX1 both in specific neuron function and endocrine mechanisms.

Objective assessment of image quality in conventional and digital mammography taking into account dynamic range.

The goal of this work is to develop a method to objectively compare the performance of a digital and a screen-film mammography system in terms of image quality. The method takes into account the dynamic range of the image detector, the detection of high and low contrast structures, the visualisation of the images and the observer response. A test object, designed to represent a compressed breast, was constructed from various tissue equivalent materials ranging from purely adipose to purely glandular composition. Different areas within the test object permitted the evaluation of low and high contrast detection, spatial resolution and image noise. All the images (digital and conventional) were captured using a CCD camera to include the visualisation process in the image quality assessment. A mathematical model observer (non-prewhitening matched filter), that calculates the detectability of high and low contrast structures using spatial resolution, noise and contrast, was used to compare the two technologies. Our results show that for a given patient dose, the detection of high and low contrast structures is significantly better for the digital system than for the conventional screen-film system studied. The method of using a test object with a large tissue composition range combined with a camera to compare conventional and digital imaging modalities can be applied to other radiological imaging techniques. In particular it could be used to optimise the process of radiographic reading of soft copy images.

Behavior of nuclear matrix proteins during camptothecin-induced apoptosis in HL-60 human leukemia cells.

In this study we focused our attention on the behavior of four nuclear matrix proteins during the various stages of apoptosis in the HL-60 cell line exposed to the DNA topoisomerase I inhibitor, camptothecin. We have examined the following antigens by immunocytochemical techniques: (i) the 180-kDa nucleolar isoform of DNA topoisomerase II; (ii) a 126-kDa polypeptide of nuclear bodies; (iii) a 125-kDa protein; and (iv) a 160-kDa polypeptide which are known to be components of the matrix inner network. Indirect immunofluorescence experiments were performed to follow these nuclear matrix antigens during apoptosis. Moreover, the ultrastructural localization of both 125- and 160-kDa proteins was investigated by electron microscope immunocytochemistry with gold-conjugated secondary antibodies. While the antibody to the nucleolar isoform of DNA topoisomerase II gave a fluorescent pattern that was well-maintained until the late phases of apoptosis, the other three nuclear antigens showed marked modifications in their distribution. A common feature, particularly evident for 125- and 160-kDa proteins, was their absence from cap-shaped chromatin marginations, whereas they were present in the areas of remaining decondensed chromatin. The 126-kDa polypeptide concentrated progressively in an irregular mass at the opposite side of the crescentic caps and then broke up in fine spots. The 125- and 160-kDa proteins localized in the nucleolus and precisely within certain granules which are known to appear in the nucleolar area after camptothecin administration. These results show that, in addition to the well-known chromatin changes, nuclear organization undergoes other rearrangements during the apoptotic process.

Macular and retinal dysfunction of unknown origin in adults with normal fundi: evidence for an autoimmune pathophysiology.

Here we report the discovery of and phenotypic characterization of a retinal disorder of unknown origin in adults using clinical, electrophysiological and psychophysical techniques, and to seek the presence of circulating retinal autoantibodies in the sera of these patients. Sixteen patients were identified with progressive bilateral visual loss over a period of months. Ten of the patients were male, and the average age was 55.3 years (range from 43 to 76 years). Known causes such as carcinoma-associated retinopathy, acute zonal occult outer retinopathy and hereditary cone dystrophy appeared unlikely. Investigations included electrophysiology, fundus autofluorescence imaging and psychophysical tests. The sera of these patients were analyzed with indirect immunocytochemistry and Western immunoblot analysis on murine (BALB/c) retinal tissue for the presence of retinal autoantibodies. Bilateral visual loss and photophobia progressed over a period of months to years (average 28.7 months, range 3-67) and subsequently stabilized. No abnormality was observed by biomicroscopy, angiography or autofluorescence imaging. Electrophysiology indicated predominant cone-system dysfunction, either macular or generalized, and post-phototransduction involvement in 9 patients (56%). Photopic and scotopic visual fields and dark adaptation kinetics showed both cone and rod system involvement in all cases. Heterogeneous immunohistochemical staining patterns were seen with the sera of these patients as compared with controls. A majority of the affected patients (9/15) stained with an antinuclear pattern. The retinal autoantibodies from the sera of most patients reacted with the retinal proteins of molecular weight between 34 and 40 kDa. The aetiology of this distinctive retinal disorder therefore appears to be mediated through an autoimmune mechanism.

An effector-reduced anti-β-amyloid (Aβ) antibody with unique aβ binding properties promotes neuroprotection and glial engulfment of Aβ.

Passive immunization against β-amyloid (Aβ) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimer's disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aβ monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aβ, protected against Aβ1-42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aβ oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP((V717I))/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aβ, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aβ antibody that takes advantage of a unique Aβ binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.

A dual functional origin of transfer in the ICEclc genomic island of Pseudomonas knackmussii B13.

Genomic islands (GEIs) are large DNA segments, present in most bacterial genomes, that are most likely acquired via horizontal gene transfer. Here, we study the self-transfer system of the integrative and conjugative element ICEclc of Pseudomonas knackmussii B13, which stands model for a larger group of ICE/GEI with syntenic core gene organization. Functional screening revealed that unlike conjugative plasmids and other ICEs ICEclc carries two separate origins of transfer, with different sequence context but containing a similar repeat motif. Conjugation experiments with GFP-labelled ICEclc variants showed that both oriTs are used for transfer and with indistinguishable efficiencies, but that having two oriTs results in an estimated fourfold increase of ICEclc transfer rates in a population compared with having a single oriT. A gene for a relaxase essential for ICEclc transfer was also identified, but in vivo strand exchange assays suggested that the relaxase processes both oriTs in a different manner. This unique dual origin of transfer system might have provided an evolutionary advantage for distribution of ICE, a hypothesis that is supported by the fact that both oriT regions are conserved in several GEIs related to ICEclc.

Molecular identification and characterization of the Arabidopsis delta(3,5),delta(2,4)-dienoyl-coenzyme A isomerase, a peroxisomal enzyme participating in the beta-oxidation cycle of unsaturated fatty acids.

Degradation of unsaturated fatty acids through the peroxisomal beta-oxidation pathway requires the participation of auxiliary enzymes in addition to the enzymes of the core beta-oxidation cycle. The auxiliary enzyme delta(3,5),delta(2,4)-dienoyl-coenzyme A (CoA) isomerase has been well studied in yeast (Saccharomyces cerevisiae) and mammals, but no plant homolog had been identified and characterized at the biochemical or molecular level. A candidate gene (At5g43280) was identified in Arabidopsis (Arabidopsis thaliana) encoding a protein showing homology to the rat (Rattus norvegicus) delta(3,5),delta(2,4)-dienoyl-CoA isomerase, and possessing an enoyl-CoA hydratase/isomerase fingerprint as well as aspartic and glutamic residues shown to be important for catalytic activity of the mammalian enzyme. The protein, named AtDCI1, contains a peroxisome targeting sequence at the C terminus, and fusion of a fluorescent protein to AtDCI1 directed the chimeric protein to the peroxisome in onion (Allium cepa) cells. AtDCI1 expressed in Escherichia coli was shown to have delta(3,5),delta(2,4)-dienoyl-CoA isomerase activity in vitro. Furthermore, using the synthesis of polyhydroxyalkanoate in yeast peroxisomes as an analytical tool to study the beta-oxidation cycle, expression of AtDCI1 was shown to complement the yeast mutant deficient in the delta(3,5),delta(2,4)-dienoyl-CoA isomerase, thus showing that AtDCI1 is also appropriately targeted to the peroxisome in yeast and has delta(3,5),delta(2,4)-dienoyl-CoA isomerase activity in vivo. The AtDCI1 gene is expressed constitutively in several tissues, but expression is particularly induced during seed germination. Proteins showing high homology with AtDCI1 are found in gymnosperms as well as angiosperms belonging to the Monocotyledon or Dicotyledon classes.

Automatic Tracer-Dilution Method Used for Stage-Discharge Ratings and Streamflow Hydrographs on Small Iowa Streams, HR-285, 1990

An automatic system was designed to concurrently measure stage and discharge for the purpose of developing stage-discharge ratings and high flow hydrographs on small streams. Stage, or gage height, is recorded by an analog-to-digital recorder and discharge is determined by the constant-rate tracer-dilution method. The system measures flow above a base stage set by the user. To test the effectiveness of the system and its components, eight systems, with a variety of equipment, were installed at crest-stage gaging stations across Iowa. A fluorescent dye, rhodamine-WT, was used as the tracer. Tracer-dilution discharge measurements were made during 14 flow periods at six stations from 1986 through 1988 water years. Ratings were developed at three stations with the aid of these measurements. A loop rating was identified at one station during rapidly-changing flow conditions. Incomplete mixing and dye loss to sediment apparently were problems at some stations. Stage hydrographs were recorded for 38 flows at seven stations. Limited data on background fluorescence during high flows were also obtained.