Oscillating viscous flow past a streamwise linear array of circular cylinders.

This paper addresses the viscous flow developing about an array of equally spaced identical circular cylinders aligned with an incompressible fluid stream whose velocity oscillates periodically in time. The focus of the analysis is on harmonically oscillating flows with stroke lengths that are comparable to or smaller than the cylinder radius, such that the flow remains two-dimensional, time-periodic and symmetric with respect to the centreline. Specific consideration is given to the limit of asymptotically small stroke lengths, in which the flow is harmonic at leading order, with the first-order corrections exhibiting a steady-streaming component, which is computed here along with the accompanying Stokes drift. As in the familiar case of oscillating flow over a single cylinder, for small stroke lengths, the associated time-averaged Lagrangian velocity field, given by the sum of the steady-streaming and Stokes-drift components, displays recirculating vortices, which are quantified for different values of the two relevant controlling parameters, namely, the Womersley number and the ratio of the inter-cylinder distance to the cylinder radius. Comparisons with results of direct numerical simulations indicate that the description of the Lagrangian mean flow for infinitesimally small values of the stroke length remains reasonably accurate even when the stroke length is comparable to the cylinder radius. The numerical integrations are also used to quantify the streamwise flow rate induced by the presence of the cylinder array in cases where the periodic surrounding motion is driven by an anharmonic pressure gradient, a problem of interest in connection with the oscillating flow of cerebrospinal fluid around the nerve roots located along the spinal canal.

Differential mechanisms of transmission at three types of mossy fiber synapse.

The axons of the dentate gyrus granule cells, the so-called mossy fibers, innervate their inhibitory interneuron and pyramidal neuron targets via both anatomically and functionally specialized synapses. Mossy fiber synapses onto inhibitory interneurons were comprised of either calcium-permeable (CP) or calcium-impermeable (CI) AMPA receptors, whereas only calcium-impermeable AMPA receptors existed at CA3 principal neuron synapses. In response to brief trains of high-frequency stimuli (20 Hz), pyramidal neuron synapses invariably demonstrated short-term facilitation, whereas interneuron EPSCs demonstrated either short-term facilitation or depression. Facilitation at all CI AMPA synapses was voltage independent, whereas EPSCs at CP AMPA synapses showed greater facilitation at -20 than at -80 mV, consistent with a role for the postsynaptic unblock of polyamines. At pyramidal cell synapses, mossy fiber EPSCs possessed marked frequency-dependent facilitation (commencing at stimulation frequencies >0.1 Hz), whereas EPSCs at either type of interneuron synapse showed only moderate frequency-dependent facilitation or underwent depression. Presynaptic metabotropic glutamate receptors (mGluRs) decreased transmission at all three synapse types in a frequency-dependent manner. However, after block of presynaptic mGluRs, transmission at interneuron synapses still did not match the dynamic range of EPSCs at pyramidal neuron synapses. High-frequency stimulation of mossy fibers induced long-term potentiation (LTP), long-term depression (LTD), or no change at pyramidal neuron synapses, interneuron CP AMPA synapses, and CI AMPA synapses, respectively. Induction of LTP or LTD altered the short-term plasticity of transmission onto both pyramidal cells and interneuron CP AMPA synapses by a mechanism consistent with changes in release probability. These data reveal differential mechanisms of transmission at three classes of mossy fiber synapse made onto distinct targets.

Long-term specification of AMPA receptor properties after synapse formation.

AMPA receptors expressed at auditory nerve synapses in the mammalian and avian cochlear nuclei display exceptionally rapid channel gating, an adaptation well suited for acoustic processing. We examined whether cellular interactions during development might determine the subunit composition of these receptors. After synapse formation in the avian nucleus magnocellularis (nMag) in vivo, the rate of receptor desensitization increased threefold, sensitivity to channel block by polyamines increased, and sensitivity to cyclothiazide, an inhibitor of desensitization, increased, indicating a reduction in glutamate receptor subunit 2 and of flip splice variants. This phenotypic switch was prevented, but not reversed, by isolating nMag neurons in a cell-culture environment. We propose that the switch in receptor kinetics is an outcome of cellular interactions during a critical period that result in the long-term determination of receptor phenotype.

Limits of flow-cytometry histogram analysis methods to assess bladder tumour antigen expression.

Tumour-associated antigens detected in cells obtained from bladder washings or tumours are useful markers in bladder cancer. Flow cytometry is commonly used to quantify immuno-stained cells. A straightforward way to analyze data is to count the fluorescent cells above a threshold empirically determined on a control histogram representation. However, specific antigens expressed at highly variable rates give rise to wide range distributions in flow cytometry as illustrated when a mucin antigen for urinary bladder was titrated by M344 monoclonal antibody in urothelial cancer cells. We have evaluated several methods of background estimation and subtraction in order to determine the proportion of M344 Mab positive cells. These include threshold setting (Histogram Shape Dependent (HSD) threshold developed in this study, 2% preset or 5% preset background), subtraction of the blank from the test histograms, and Kolmogorov-Smirnov statistical test. The HSD method appeared to be a more reliable method for background estimation; however, in the case of very low antigen expression, where specific fluorescence histograms could hardly be distinguished from that of the background, fluorescence microscopy remained the only valid method, since it allowed the distinction between specific and non-specific fluorescence on the basis of structural differences between the two.

One- and two-dimensional histone separations in acidic gels: usefulness of methylene blue-driven photopolymerization.

We tested the recently introduced methylene blue-toluene sulfinate-diphenyl-iodonium polymerization system in order to prepare acetic acid-urea-Triton X-100 gels for histone separations. When compared to standard persulfate-based initiators, this system exhibited several advantages. First, the polymerization proceeds at a much faster rate but is easily controlled since it is light-dependent. Second, no prerunning of the gel was required, since there is no oxidizing molecule able to introduce artifacts. Moreover, this procedure produces gels presenting cleaner backgrounds with silver staining. The ability of the procedure to carry out high resolution two-dimensional electrophoresis of histones was also examined. High resolution two-dimensional gels, using photopolymerized acidic gels as the first or second dimension were obtained.

Early events in erythroid differentiation: accumulation of the acidic peroxidoxin (PRP/TSA/NKEF-B).

The acidic peroxidoxin [also named thiol-specific antioxidant protein (TSA) or protector protein (PRP)], which plays a role in the response against oxidative stress, is one of the major proteins of red blood cells. In this work, we show that this protein is induced at early stages of erythroid differentiation prior to haemoglobin accumulation, which suggests that it may play a role at the erythroblast stage, where haemoglobinized, nucleated and genetically active cells are submitted to a maximally dangerous oxidative stress. The early accumulation of this protein has been demonstrated both on transformed cell systems and on normal differentiating human erythroid cells. This suggests that this protein may play an important role in the differentiation of the erythroid cells.

Induction of stathmin expression during erythropoietic differentiation.

Stathmin is a M(r) 19,000 cytosolic protein proposed to act as a relay for signal-activating pathways regulating cell proliferation and differentiation. In a study of erythropoietic differentiation, stathmin was detected as a protein that is induced during the early stages of differentiation in several cellular model systems. The unphosphorylated form of stathmin was most prominently induced, which suggests that this form does not only play a role in pathways oriented toward cell proliferation, as is the case for lymphocytic systems, but may also play a role in various differentiation pathways.

Developmentally regulated chromatin acetylation and histone H1(0) accumulation.

There exists a close relationship between core histone acetylation and the induced expression of the histone H1(0) gene. We took advantage of this fact to evaluate the influence of chromatin hyperacetylation on the developmentally regulated expression of this specific gene. In this study, the in situ immunodetection approach has been used to analyze both the acetylated histone H4 isoforms and histone H1(0) accumulation during early Xenopus laevis development. We have chosen two stages of development, gastrula stage, when H1(0) is not expressed and not inducible by butyrate treatment, and stage 27 when H1(0) is not expressed but is inducible by butyrate. At stage 27 of development, the early induced accumulation of histone H1(0) under butyrate treatment, occurs mainly in tissues that express the protein normally during later development. These experiments suggest that histone acetylation may be part of a pathway which, in a specific set of cells, keeps H1(0) and probably a series of specific genes, competent for transcription, but cell-specific factors are involved in the induced expression of these genes.

Accumulation of histone H1(0) during early Xenopus laevis development.

It is known that a transition in the linker-histone variants takes place within chromatin during early development of Xenopus laevis; a cleavage-type H1 is replaced by the somatic type. Based on cytofluorimetric analysis of the distribution of the embryo cells in the cell cycle, we showed that this previously described transition occurs when significant modifications of the proliferative capacities of the cells occur. Moreover, this analysis allowed us to show that cell proliferation decreases gradually after the gastrula stage of development. This period terminates with the arrest of more than 90% of cells in the G0/G1 phase of the cell cycle at stage 45. We showed that the major accumulation of the differentiation-specific H1 subtype, histone H1(0), occurs at this time. H1(0), first detected in a restricted set of tissues, is then widely expressed during the later development at stage 45. Moreover, the double staining of nuclei isolated from embryo cells, for H1(0) and DNA, allowed us to show that this accumulation of H1(0) is not restricted to arrested cells. The example of the Xenopus early development shows that there may be an adaptation of the type of H1 expressed to the proliferative abilities of cells. This observation may provide insight into the significance of the expression of different H1 subtypes during development.

Variation of H1(0) content throughout the cell cycle in regenerating rat liver.

Histone H1(0), a differentiation-specific member of the histone H1 family, accumulates in cells during the terminal phase of cell differentiation, in tissues composed of arrested cells or cells exhibiting little proliferation. Moreover, the induction of cell proliferation in vivo, i.e., after partial hepatectomy, is accompanied by a decrease in H1(0) content. These observations suggest that H1(0) may be involved in the arrest of cell proliferation in vivo. In order to investigate this possibility, we took advantage of the fact that after partial hepatectomy the initiation of cell division is not synchronous. The strategy was to know, at the level of a single cell, whether H1(0) decreases prior to the initiation of the S phase or whether a cell can initiate DNA replication having a significant amount of H1(0) in the nucleus. We defined new protocols to analyze H1(0) content and cell proliferation at the level of a single cell, both in situ and by flow cytometry. The simultaneous determination of the relative amount of H1(0) and the position of cells in the cell cycle showed that no significant difference in H1(0) content was detected in cells actively replicating their DNA compared to nondividing cells. These observations have been confirmed by the successive immunodetections of H1(0) and BrdU in situ on the same cells. Therefore, we show here that in vivo, cells can initiate DNA replication with significant amounts of H1(0) and that the decrease of H1(0) is not a prerequisite of cell division. We propose that the accumulation of H1(0) is not related to the arrest of cell proliferation, but is controlled in such a manner that the protein accumulates in slowly dividing cells and decreases in rapidly growing cells.

Pharmacological characterization of heterodimeric NMDA receptors composed of NR 1a and 2B subunits: differences with receptors formed from NR 1a and 2A.

Pharmacological and molecular biological evidence indicates the existence of multiple types of NMDA receptors within the CNS. We have characterized pharmacological properties of receptors assembled from the combination of NR 1a and NR 2B subunits (NR 1a/2B) expressed in transfected cells using both 125I-MK-801 binding assays and electrophysiological measures. Binding of 125I-MK-801 to cells transfected with NR 1a/2B is saturable with a KD of 440 pM. The binding is potently inhibited by ketamine, dextromethorphan, phencyclidine, and MK-801 and is stimulated by low concentrations of magnesium. These properties resemble those of native receptors and receptors produced by NR 1a/2A. However, 125I-MK-801 binding to membranes from cells transfected with NR 1a/2B is inhibited with high affinity by ifenprodil and is stimulated by spermidine, unlike receptors assembled from NR 1a/2A. NMDA-induced currents measured in cells transfected with either NR 1a/2A or NR 1a/2B have pharmacological properties that correlate well with the binding studies. Currents in cells transfected with NR 1a/2B are potentiated by spermidine and blocked with high affinity by ifenprodil, whereas currents in cells transfected with NR 1a/2A are not enhanced by spermidine and are weakly inhibited by ifenprodil. These data suggest that pharmacological heterogeneity in native NMDA receptors may be explained by combinations of different subunits.

Sample application by in-gel rehydration improves the resolution of two-dimensional electrophoresis with immobilized pH gradients in the first dimension.

We describe a modification in the sample application mode for isoelectric focusing with immobilized pH gradients. Instead of being applied at the surface of the gel in a sample cup, the sample is introduced into the gel during the immobilized pH gradient strip rehydration step. This modification implies the use of low percentage gels (below 3.5% T) and specially designed, but simple, rehydration chambers. The main advantages are a uniform resolution without side effects and the possibility of handling large sample volumes (500 microL for a standard 3 x 160 x 0.5 mm strip), allowing micropreparative work (milligram samples) with a simple experimental design.

Relationship between core histone acetylation and histone H1(0) gene activity.

In this study we show a striking correlation between histone H1(0) gene expression and histone acetylation. Trichostatin A, a highly specific inhibitor of histone deacetylase, efficiently induces H1(0) gene expression. Moreover, using a cell line sensitive to trichostatin A (FM3A) and a derived cell line selected for its resistance to this inhibitor (TR303), it is shown that the level of H1(0) gene expression is related to the extent of chromatin acetylation. After showing the S-phase-dependent activation of H1(0) gene expression, we demonstrate that hyperacetylation has a dominant effect on H1(0) gene expression, since it enhances the expression of the gene independent of the position of cells in the cell cycle. This response to deacetylase inhibitors is specific to H1(0), since it is not shared by other cell-cycle-dependent histone genes (H1 and H4). Finally, by transfection of trichostatin-A-resistant and trichostatin-A-sensitive cells with a plasmid containing a H1(0) promoter, we show that the exogenous H1(0) promoter is also highly sensitive to trichostatin A treatment and that activation of transcription follows exactly the same pattern as activation of the endogenous gene. These data show that histone acetylation may be used to modulate H1(0) gene activity and offers insight into a possible mechanism in which the developmentally regulated chromatin acetylation acts to potentiate H1(0) gene expression.

Molecular basis of the activation of basal histone H1(0) gene expression.

Histone H1(0) is encoded by a gene that is expressed only in cells committed to differentiation. We have previously cloned the Xenopus laevis H1(0) gene and studied elements involved in the regulation of its expression in transfected Xenopus laevis A6 cells, and in microinjected embryos. In this work, in order to understand the basis of the action of these elements, we used an A6 cell nuclear extract and showed that the H1(0) promoter is able to direct efficient in vitro transcription, which is highly dependent on a functional TATA box. However, in contrast to what we observed in vivo, in transfected A6 cells, the in vitro transcription was independent of major regulatory elements, defined in vivo. We then used this in vitro system to reconstitute H1(0) gene regulation. The creation of a repressive environment by the addition of purified histone H1 to the in vitro transcription system allowed us to obtain transcription dependent on the integrity of the regulatory elements. Investigating the basis of this regulation we found that protein-DNA interaction on the proximal promoter region was dependent on the integrity of proximal elements, and moreover the distal regulatory element, the UCE, was able to modulate this interaction. We conclude that the role of these regulatory elements is to maintain the basal TATA-dependent transcription of H1(0) under repressive condition: i.e., H1-mediated repression of transcription, or chromatin assembly in general.

Characterization of baculovirus recombinant wild-type p53. Dimerization of p53 is required for high-affinity DNA binding and cysteine oxidation inhibits p53 DNA binding.

A high-yield, rapid and non-denaturing purification protocol for baculovirus recombinant wild-type p53 is described. Gel-filtration chromatography and chemical cross-linking experiments indicated that purified p53 assembles into multimeric forms ranging from tetramer to higher oligomers. A gel-mobility-shift assay and protein-DNA cross-linking studies demonstrated that purified baculovirus recombinant p53 binds to consensus DNA target as a dimer but that additional p53 molecules may then associate with the preformed p53-dimer-DNA complexes to form larger p53 DNA complexes. These observations suggest that the p53 tetramers and higher oligomers that form the minimal p53 association in solution dissociate upon DNA binding to form p53 dimer-DNA complexes. Binding of the mAB PAb 421 to the oligomerization-promoting domain on p53 stimulated sequentially formation of both p53-dimer-DNA and larger p53-DNA complexes. This observation suggests that factors may exist in vivo that could participate in the formation and the stabilization of the various p53-DNA complexes. Further characterization of the purified p53 revealed that the protein possesses highly reactive cysteine residues. We show that intrachain disulfide bonds form within the purified p53 molecules during storage in the absence of reducing agent. Zn2+ binding to p53 protect sulfhydryl groups from oxidation. Cysteine oxidation by intramolecular disulfide-bond formation did not modify the wild-type immunoreactive phenotype of the p53 protein but totally inhibited its DNA-binding activities. The oxidation of the p53 cysteine residues was also observed for nuclear p53 in baculovirus-infected insect cells. The redox status of the nuclear p53 regulates its DNA-binding activity in vitro confirming the essential role of the reduced state of cysteine residues in p53 for detectable DNA-binding activity.

Two mRNA species encoding the differentiation-associated histone H1(0) are produced by alternative polyadenylation in mouse.

Histone H1(0) is a differentiation-specific member of the histone H1 family. The accumulation of the protein is associated with the terminal stage of cell differentiation and is regulated at various levels. In mouse, the analysis of the expression of the single copy gene encoding H1(0) has shown that another H1(0)-related mRNA species (0.9 kb) is present in addition to the usual 2.1-kb mRNA. In this study, we have cloned and sequenced the smaller H1(0)-related mRNA. This mRNA seems to be produced by the use of an additional polyadenylation signal present in the 3' untranslated region (UTR) of the initial transcript. This smaller H1(0)-encoding mRNA is expressed only in mouse and is transferred into polysomes as efficiently as the larger version upon the induction of cell differentiation. The use of the described polyadenylation site removes over 1 kb of the 3' UTR of H1(0) mRNA and seems to be involved in the regulation of H1(0) mRNA stability.

Silver-staining of proteins in polyacrylamide gels: a general overview.

On the basis of the physico-chemical principles underlying silver-staining of proteins, which are recalled in this paper, several methods of silver-staining of proteins after SDS electrophoresis in polyacrylamide gels or isoelectric focusing were tested. The most valuable protocols are presented in this report, including standard methods for unsupported gels and new methods devised for thin (0.5 mm) supported gels for SDS electrophoresis or isoelectric focusing and for staining of small peptides. Generally speaking, the most rapid methods were found to be less sensitive and less reproducible than more time-consuming ones. Among the long methods, those using silver-diammine complex gave the most uniform sensitivity. They require however special home-made gels and cannot be applied to several electrophoretic systems (e.g. systems using tricine or bicine as the trailing ion, or isoelectric focusing in immobilized pH gradients). For these reasons, protocols based on silver nitrate are of a more general use and might be favored. Future trends for silver-staining will also be discussed.

Flow and image cytometry for DNA analysis in bladder washings: improved concordance by using internal reference for flow.

Using flow or image cytometry, we compared the DNA distribution of cells from bladder washings from 52 patients with bladder cancer. For image cytometry, urothelial nuclei (recognized visually) were analyzed for DNA content using polymorphonuclear nuclei as internal diploid reference. For flow cytometry, two methods can be used: either all cells can be analyzed, as commonly performed, or urothelial cells can be analyzed alone, after specific detection. In this flow cytometry study, cells were doubly stained for panurothelial antigens T16 and for DNA. All the cells were first analyzed using peripheral lymphocytes as an external reference; 79% of the results were similar with results obtained from image analysis. For discordant results, flow-cytometric data were reprocessed to identify immunologically stained urothelial cells; one additional case became concordant with image cytometry when only urothelial cells were analyzed, with lymphocytes as diploid reference; a better concordance (94%) was found when the nonurothelial cells of the samples served as a diploid reference instead of peripheral lymphocytes. This suggests that we achieved an improvement of the flow-cytometric evaluations for ploidy assessment, and we conclude that, on these conditions, flow or image analysis can be considered as equivalent methods for DNA content studies of tumors.

Selective cytotoxicity of L-glutamic acid gamma-monohydroxamate (GAH) for melanoma tumor cells.

We have previously shown that L-glutamic acid gamma-monohydroxamate (GAH) exhibits an antitumor activity, both in vitro and in vivo. In this report we explore the selective cytotoxicity of GAH in vitro by comparing the survival of tumor and normal cells. GAH exerts an irreversible delayed effect with tumoral cells and a reversible effect with normal cells: after a short incubation time of 6 hrs in the presence of 1.2 mM GAH and after removal of the drug, the survival of N Ter Dau and MRC5 cells was identical reaching about 85% after 24 hrs of culture. But, after another 48 hrs of culture, MRC5 cells recovered 100% cell survival while with N Ter Dau cells the survival decreased to 65%. A longer exposure time to GAH (18 hrs) and an additional 54 hrs of culture after removal of GAH led to 50 +/- 10% of cell survival with normal cells but only 25 +/- 10% with tumor cells. Using a long-term clonogenic assay, we showed that the 25% N Ter Dau cells surviving at 72 hrs after GAH treatment led mainly to abortive colonies (17% +/- 3%) with only 2.3 +/- 0.9% of surviving colonies. Such a difference does not exist for normal cells. Cell cycle analysis of tumor and normal cells treated with GAH (18 hrs, 1,2 mM) has shown that the drug prevents both cell type from cycling from G1 to S phase. However, the two cell types started to cycle again after removal of GAH but a delay of 24 hrs was observed for tumoral cells compared to normal cells.

Light and electron microscope immunocytochemical analyses of histone H1(0) distribution in the nucleus of Friend erythroleukemia cells.

The localization of histone H1(0) in murine erythroleukemia cells which were induced to resume a differentiation program was studied in cells which have recovered their proliferative capacity after transient blockage in the G1 phase of the cell cycle. Previous studies have shown that histone H1(0) accumulation occurs at early times of induction and is probably related to the commitment itself. The distribution of the protein was determined by immunomicroscopy with monoclonal antibodies specific for histone H1(0). Our observations showed that the histone accumulates in nuclei. Immunoelectron microscopy further demonstrated the presence of histone H1(0) in condensed chromatin areas, including perinucleolar chromatin. Moreover, histone H1(0) also occurred in the perichromatin regions, previously described as preferential sites of pre-mRNA synthesis, suggesting that histone H1(0) is not fully excluded from active chromatin.