5 research outputs found
Activation of p34cdc2 protein kinase by microinjection of human cdc25C into mammalian cells. Requirement for prior phosphorylation of cdc25C by p34cdc2 on sites phosphorylated at mitosis.
International audienceHuman cdc25C protein, a specific tyrosine phosphatase that activates the p34cdc2 protein kinase at mitosis, is itself a phosphoprotein that shows increased phosphorylation during the G2-M transition. In vitro, cdc25C protein is substantially phosphorylated by purified p34cdc2-cyclin B protein kinase. Of seven putative phosphorylation sites for p34cdc2 protein kinase present in human cdc25C, five are phosphorylated by p34cdc2 protein kinase in vitro, as assessed by tryptic phosphopeptide mapping and peptide sequencing. These same sites are also phosphorylated in vivo during the G2-M transition in normal mammalian fibroblasts and have been precisely mapped. The cdc25C phosphorylated in vitro by p34cdc2 protein kinase exhibits a 2-3-fold higher activity than the nonphosphorylated cdc25C, as assayed by activation of inactive cdc2 prokinase. Microinjection of purified cdc25C proteins into living fibroblasts reveals that only the phosphorylated form of cdc25 is highly effective in activating G2 cells into premature prophase in a manner similar to microinjection of purified active p34cdc2 protein kinase. Together these data show that multisite phosphorylation of cdc25C by p34cdc2-cyclin B protein kinase occurs at the G2-M transition and is sufficient to induce the autoamplification of cdc2/M-phase promoting factor necessary to drive somatic mammalian cells into mitosis
Cold-induced changes in amphibian oocytes.
International audienceFemale Pleurodeles waltl newts (Amphibia, urodele), usually raised at 20 degrees C, were submitted to low temperatures; oocytes responded to this cold stress by drastic changes both in lampbrush chromosome structure and in protein pattern. Preexisting lateral loops of lampbrush chromosomes were reduced in size and number, while cold-induced loops which were tremendously developed, occurred on defined bivalents of the oocyte at constant, reproducible sites. A comparison of protein patterns in control and stressed oocytes showed two main differences: in stressed oocytes, overall protein synthesis was reduced, except for a set of polypeptides, the "cold-stress proteins"; second, there was a striking inversion of the relative amount of beta- and gamma-actin found in the oocyte nucleus before and after cold stress. Whereas beta-actin was the predominant form in control oocytes, gamma-actin became the major form in stressed oocytes
Interaction of Antibiotics with Lipid Vesicles on Thin Film Porous Silicon Using Reflectance Interferometric Fourier Transform Spectroscopy
International audienceThe ability to observe interactions of drugs with cell membranes is an important area in pharmaceutical research. However, these processes are often difficult to understand due to the dynamic nature of cell membranes. Therefore, artificial systems composed of lipids have been used to study membrane properties and their interaction with drugs. Here, lipid vesicle adsorption, rupture, and formation of planar lipid bilayers induced by various antibiotics (surfactin, azithromycin, gramicidin, melittin and ciprofloxacin) and the detergent dodecyl-b-D-thiomaltoside (DOTM) was studied using reflective interferometric Fourier transform spectroscopy (RIFTS) on an oxidized porous silicon (pSi) surface as a transducer. The pSi transducer surfaces are prepared as thin films of 3 μm thickness with pore dimensions of a few nanometers in diameter by electrochemical etching of crystalline silicon followed by passivation with a thermal oxide layer. Furthermore, the sensitivity of RIFTS was investigated using three different concentrations of surfactin. Complementary techniques including atomic force microscopy, fluorescence recovery after photobleaching, and fluorescence microscopy were used to validate the RIFTS-based method and confirm adsorption and consequent rupture of vesicles to form a phospholipid bilayer upon the addition of antibiotics. The method provides a sensitive and real-time approach to monitor the antibiotic-induced transition of lipid vesicles to phospholipid bilayers
Interaction of Antibiotics with Lipid Vesicles on Thin Film Porous Silicon Using Reflectance Interferometric Fourier Transform Spectroscopy
The ability to observe interactions
of drugs with cell membranes
is an important area in pharmaceutical research. However, these processes
are often difficult to understand due to the dynamic nature of cell
membranes. Therefore, artificial systems composed of lipids have been
used to study membrane properties and their interaction with drugs.
Here, lipid vesicle adsorption, rupture, and formation of planar lipid
bilayers induced by various antibiotics (surfactin, azithromycin,
gramicidin, melittin and ciprofloxacin) and the detergent dodecyl-<i>b</i>-d-thiomaltoside (DOTM) was studied using reflective
interferometric Fourier transform spectroscopy (RIFTS) on an oxidized
porous silicon (pSi) surface as a transducer. The pSi transducer surfaces
are prepared as thin films of 3 μm thickness with pore dimensions
of a few nanometers in diameter by electrochemical etching of crystalline
silicon followed by passivation with a thermal oxide layer. Furthermore,
the sensitivity of RIFTS was investigated using three different concentrations
of surfactin. Complementary techniques including atomic force microscopy,
fluorescence recovery after photobleaching, and fluorescence microscopy
were used to validate the RIFTS-based method and confirm adsorption
and consequent rupture of vesicles to form a phospholipid bilayer
upon the addition of antibiotics.
The method provides a sensitive and real-time approach to monitor
the antibiotic-induced transition of lipid vesicles to phospholipid
bilayers
Characterization of Phospholipid Bilayer Formation on a Thin Film of Porous SiO<sub>2</sub> by Reflective Interferometric Fourier Transform Spectroscopy (RIFTS)
Classical methods for characterizing supported artificial
phospholipid bilayers include
imaging techniques such as atomic force microscopy and fluorescence
microscopy. The use in the past decade of surface-sensitive methods
such as surface plasmon resonance and ellipsometry, and acoustic sensors
such as the quartz crystal microbalance, coupled to the imaging methods,
have expanded our understanding of the formation mechanisms of phospholipid
bilayers. In the present work, reflective interferometric Fourier
transform spectrocopy (RIFTS) is employed to monitor the formation
of a planar phospholipid bilayer on an oxidized mesoporous Si (pSiO<sub>2</sub>) thin film. The pSiO<sub>2</sub> substrates are prepared
as thin films (3 μm thick) with pore dimensions of a few nanometers
in diameter by the electrochemical etching of crystalline silicon,
and they are passivated with a thin thermal oxide layer. A thin film
of mica is used as a control. Interferometric optical measurements
are used to quantify the behavior of the phospholipids at the internal
(pores) and external surfaces of the substrates. The optical measurements
indicate that vesicles initially adsorb to the pSiO<sub>2</sub> surface
as a monolayer, followed by vesicle fusion and conversion to a surface-adsorbed
lipid bilayer. The timescale of the process is consistent with prior
measurements of vesicle fusion onto mica surfaces. Reflectance spectra
calculated using a simple double-layer Fabry–Perot interference
model verify the experimental results. The method provides a simple,
real-time, nondestructive approach to characterizing the growth and
evolution of lipid vesicle layers on the surface of an optical thin
film