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At least 433 records · Page 24Linked to original sources
PubMed1994

Cold-induced conformational changes of ribonuclease A as investigated by subzero transverse temperature gradient gel electrophoresis.

Subzero temperature gradient gel electrophoresis is a new approach which allows to measure the transition temperature of low temperature-induced subtle conformational changes of proteins and to detect the different conformational states, including unfolded states. Using this technique under destabilizing conditions, i.e., in the presence of 4 M urea, bovine pancreas ribonuclease A exhibited two transitions: (i) a continuous transition with a midpoint temperature of -14 degrees C corresponding to a rapid equilibrium between the initial enzyme state and a conformational state more compact than the initial one; (ii) a discontinuous transition at -22.5 degrees C from intermediate to a non migrating species. Under reducing conditions this second transition was shifted toward high temperatures (-18.5 degrees C). We attempted to detect these two transitions by differential scanning calorimetry, UV spectrophotometry and circular dichroism measurements. These transitions have been ascribed to subtle cold-induced conformational changes.

Calorimetry, Differential Scanning↗
PubMed1995

A sensitive double immunostaining protocol for Fos-immunoreactive neurons.

Immunostaining of Fos, the nuclear protein encoded by the immediate early gene c-fos, is widely used to reveal the functional activation of neurons. The chemical identity of cells that express c-fos can be investigated with double immunohistochemistry. We report the usefulness of a sequential two-color avidin-biotin-immunoperoxidase method that provides a highly sensitive double immunostaining and allows long-term storage of the sections. In this protocol, metal intensification of diaminobenzidine (int-DAB) resulted in dark brown/black Fos immunostaining of the neuronal nucleus. The use of alpha-naphthol/pyronin reaction product yielded pink immunostaining of a second antigen in the cytoplasm. This combination produced higher contrast than that produced by int-DAB Fos immunostaining combined with conventional DAB light brown cytoplasmic staining. The sensitivity of the use of int-DAB and alpha-naphthol/pyronin was verified in different experimental paradigms, combining the immunocytochemical detection of Fos with that of the p75 nerve growth factor receptor, or parvalbumin, or calbindin D28k.

Ammonium Sulfate↗
PubMed1994

Studies on inactivation of pathogenic microorganisms in culture media and in bovine semen by photosensitive agents.

The application of three photosensitive agents for disinfection of bovine semen was investigated. Bovine microbial pathogens suspended in tissue culture medium and/or PBS and also added to bovine semen were exposed to the photosensitive agents followed by irradiation. Hematoporphyrin, hematoporphyrin derivative and thiopyronine were effective against bovine herpes virus-1, bovine viral diarrhoea virus, Mycoplasma bovigenitalium, Mycoplasma canadense, and Ureaplasma diversum in culture media. In addition, thiopyronine was effective against Leptospira pomona. Similar treatments were not effective against Leptospira hardjo, Mycoplasma bovis, or Campylobacter fetus subsp. venerealis. When microorganisms were added to bovine semen, only bovine herpes virus-1 was controlled by the photosensitive agents used at concentrations which did not appear harmful to sperm cells.

Animals↗
PubMed2004

Electroporation of cell membranes supporting penetration of photodynamic active macromolecular chromophore dextrans.

The aim is to demonstrate that macromolecular chromophore dextrans (Cibacron-dextran) acting as photosensitizers can be transported easily into cancer cells by electroporation of their membranes (short electric pulses on cell suspension between electrodes). There are two possibilities, either:(A)irradiation starts with the electropulse-showed with easily penetrating thiopyronin-yielding nearly 100% dead cells;(B)irradiation starts after a resealing time of membrane pores during which macromolecular photosensitizers can penetrate into cells. In this way, fractions of Cibacron-dextran with molecular weights (Mw) 3300, 10,900 and 500,000 are now able to kill. This combination of bioelectrochemistry and photobiology will be suitable also for other biopolymers, connected with photodynamic active chromophores (e.g. chromopeptides) to transport them through cell walls and membranes into cells and tissues. The human cancer cells U-935 and K-562 (pulsed by 1.15 kV/cm field strength) additionally or synergistically reach high rates of necrotic cells (colored by trypan blue) by this combination.

Cell Line, Tumor↗
PubMed2004

Polycomb group gene mel-18 modulates the self-renewal activity and cell cycle status of hematopoietic stem cells.

OBJECTIVE: Mel-18 is a member of the mammalian Polycomb group (PcG) genes. This family of genes regulates global gene expression in many biologic processes, including hematopoiesis and anterior-posterior axis formation by manipulating specific target genes, including members of the Hox family. Here, we demonstrate that mel-18 negatively regulates the self-renewal activity of hematopoietic stem cells (HSCs). MATERIALS AND METHODS: Long-term reconstitution activity was evaluated by competitive repopulating unit (CRU) and mean activity of the stem cells (MAS) assays in vivo in bone marrow cells (BMCs) derived from mel-18(-/-) and mel-18 tg mice. The expression levels of mel-18 and Hoxb4 were measured by quantitative real-time reverse transcription polymerase chain reaction. RESULTS: The Hoxb4 gene was highly expressed in HSCs derived from mel-18(-/-) mice. The observed CRUs were 3.21, 4.77, 3.32, and 1.64 CRU per 10(5) BMCs in mel-18(+/+), mel-18(-/-), C57BL/6, and mel-18 tg, respectively. MAS was 0.58, 0.18, 0.41, and 5.89 in mel-18(+/+), mel-18(-/-), C57BL/6, and mel-18 tg, respectively. The percentage in G0 phase HSCs (lin(-)flk2(-)c-Kit(+)Sca1+ cells) was increased in mel-18(-/-) mice and decreased in mel-18 tg mice. CONCLUSION: Loss or knockdown of mel-18 leads to the expression of Hoxb4, an increase in the proportion of HSCs in G0 phase, and the subsequent promotion of HSC self-renewal. These findings will enable us to develop new approaches for controlling HSC activity for hematopoietic transplantations based on ex vivo expansion of HSCs.

Animals↗
PubMed1997

Prediction of growth sensitivity of acute promyelocytic leukemia cells to granulocyte colony-stimulating factor using 7AAD/PY during administration of all-trans retinoic acid.

We discussed utility of cell cycle and phenotypic analysis of acute promyelocytic leukemia (APL) cells using 7AAD/PY for the prediction of efficacy and risks of all-trans retinoic acid (ATRA) and granulocyte colony-stimulating factor (G-CSF) administration to patients with APL. Serial changes in phenotype and cell kinetics of APL cells from two patients were analyzed during ATRA administration. CD15 and CD11b were expressed on the APL cells in vivo as neutrophil maturation markers, while growth activity of the cells was decreased during ATRA administration. Using 7AAD/PY, changes in phenotype and cell kinetics were clearly detected after 2 days of cultivation with ATRA and/or G-CSF. In one patient, APL cells harvested from marrow during the first 3 weeks of ATRA administration showed distinct growth sensitivity to G-CSF ex vivo, and the cells harvested after a 4-week exposure to ATRA appeared to have lost this sensitivity. In this patient, G-CSF could be safely administered after 4 weeks of ATRA therapy. 7AAD/PY analysis is useful for predicting growth sensitivity of APL cells to G-CSF during ATRA administration.

Adult↗
PubMed1998

Improved cytochemical methods for demonstrating cell death using LR White as an embedding medium.

A series of techniques based on LR White resin are described, which permit the use of an anti-histone antibody for the in situ localization of DNA fragmentation characteristic of apoptosis at both the light and the electron microscope level. The methods, applied to an untreated squamous carcinoma of the pharynx, allow direct comparison of light microscopic localization of exposed nucleosomal histones using 3,3'-diaminobenzidine (DAB) and silver-enhanced techniques with a colloidal gold-based anti-histone technique at the electron microscope level. Parallel histochemical localization of acid phosphatase activity is also presented.

Acid Phosphatase↗
PubMed2004

Bcl-2 inhibition of T-cell proliferation is related to prolonged T-cell survival.

Bcl-2 promotes oncogenesis by inhibiting cell death. Bcl-2 also inhibits proliferation and suppresses tumorigenesis in some settings. To clarify the role of the antiproliferative function of Bcl-2, mice expressing a mutant form of Bcl-2 reported to lack antiproliferative activity were generated (tyrosine 28 to alanine, Bcl-2-Y28A). As expected, both wild type (WT) and Bcl-2-Y28A inhibited apoptosis similarly. In contrast to previous results in cell lines, Bcl-2-Y28A inhibited T-cell proliferation identical to WT-Bcl-2. Significantly, both Bcl-2-Y28A and WT-Bcl-2 inhibited proliferation of T cells isolated from older animals, but not proliferation of T cells from immature mice. Instead, inhibition of cell activation correlated with T-cell size, p27 levels, and RNA content, all indicators of quiescent G0 arrest. Consistent with this model, Bcl-2 inhibition of T-cell proliferation was reversed by expression of Bax, again correlating cell proliferation with cell size. These experiments do not support genetically separate effects of Bcl-2 on apoptosis and proliferation. Instead, the data support a model in which Bcl-2 and Bax regulate T-cell proliferation by changes in T-cell size and by increasing the markers of quiescent G0 arrest. These changes likely result from prolonged T-cell survival.

Age Factors↗
PubMed1996

Photo-induced inactivation of viruses: adsorption of methylene blue, thionine, and thiopyronine on Qbeta bacteriophage.

The adsorption of cationic organic dyes (methylene blue, thionine, and thiopyronine) on Qbeta bacteriophage was studied by UV-visible and fluorescence spectroscopy. The dyes have shown a strong affinity to the virus and some have been used as sensitizers for photo-induced inactivation of virus. In the methylene blue concentration range of 0.1-5 microM and at high ratios of dye to virus (greater than 1000 dye molecules per virion), the dyes bind as aggregates on the virus. Aggregation lowers the efficiency of photoinactivation because of self-quenching of the dye. At lower ratios of dye to virus (lower than 500 dye molecules per virion), the dye binds to the virus as a monomer. Fluorescence polarization and time-resolved studies of the fluorescence support the conclusions based on fluorescence quenching. Increasing the ionic strength (adding NaCl) dissociates bound dye aggregates on the virus and releases monomeric dye into the bulk solution.

Adsorption↗
PubMed1978

Photodynamic activity of dyes with different DNA binding properties I. free-radical induction in DNA.

The photosensitizing efficiencies of eight dyes have been compared; two acridines, two xanthene derivatives, one sulphur-containing dye and three chemotherapeutic agents. The analysed reaction was the photosensitized induction of free radicals in calf-thymus DNA at low temperature. The binding of these dyes to DNA was first measured. Both strong (process I) and weak (process II) binding, with different intensities, either alone or together, were observed as mode of fixation. Whatever the nature of their binding, all the dyes used revealed a photosensitizing power as inducers of peroxide radicals in DNA. Their relative efficiencies, expressed as a function of the amount of dye molecules bound to DNA, were found to be very different. Intercalation, however, appeared to favour the free-radical induction as the first strongly bound molecules were more efficient.

Acridines↗
PubMed1987

Thiopyronine-sensitized photodynamic effect on cell growth, RNA and DNA synthesis of Chinese hamster ovary cells.

After treatment of Chinese hamster ovary (CHO) cells with very low concentrations of thiopyronine (TP; 1 microgram/ml) and visible light, a delay in growth of cell cultures (prolongation of the lag phase] was observed. The lengthened lag phase, however, was followed by normal growth of the cells. The length of the lag period is dependent on the irradiation dose applied. A similar effect on DNA and RNA synthesis could be seen after photodynamic treatment with TP in CHO cells: the maxima of RNA and DNA synthesis occur later but are not significantly reduced after treatment with low concentrations of TP and irradiation with visible light. This result is further evidence that the photodynamic effect with TP does not involve attack on nuclear DNA in eukaryotic cells.

Animals↗
PubMed2005

Effect of 1-(1-naphthylmethyl)-piperazine, a novel putative efflux pump inhibitor, on antimicrobial drug susceptibility in clinical isolates of Enterobacteriaceae other than Escherichia coli.

OBJECTIVES: 1-(1-Naphthylmethyl)-piperazine (NMP) has been shown to reverse multidrug resistance (MDR) in Escherichia coli overexpressing RND type efflux pumps, but there is no data on its activity in Enterobacteriaceae other than E. coli. METHODS: The antimicrobial susceptibilities of laboratory strains and 167 clinical isolates of Enterobacteriaceae to a variety of antimicrobial agents were determined in the absence and presence of NMP and, for comparison, of Phe-Arg-beta-naphthylamide (PAbetaN), another putative efflux pump inhibitor (EPI). A 4-fold or greater reduction of the MIC after EPI addition was considered significant. RESULTS: NMP consistently reduced the MIC of linezolid in Citrobacter freundii, Enterobacter aerogenes and Klebsiella pneumoniae clinical isolates. Significant effects of NMP addition in >50% of tested isolates were also seen for levofloxacin, tetracycline and chloramphenicol in E. aerogenes, and for levofloxacin and tetracycline in K. pneumoniae, whereas no or minor effects were observed in Serratia marcescens. MDR reversal by NMP was more likely in isolates with decreased susceptibility to fluoroquinolones. In most fluoroquinolone-resistant strains the activity was sufficient to render isolates drug-susceptible at clinically achievable concentrations. The activity of PAbetaN was different from that of NMP, suggesting different modes of action of the two putative EPIs. CONCLUSION: NMP has moderate activity in reversing MDR in many but not all members of the Enterobacteriaceae family including clinical isolates. Its effects on resistance reversal depend on bacterial species and drug, and are different from those seen with PAbetaN.

Anti-Bacterial Agents↗