Electrotransformation studies in Clostridium cellulolyticum

Electropermeabilization of Clostridium cellulolyticum was optimized using ATP leakage assays. Electrotransformation was then performed under optimized conditions (6 to 7.5 kV cm⁻¹ field strength applied during 5 ms to a mixture containing methylated plasmids and late exponential phase cell suspensions (10 molecules:1 cell) in a sucrose-containing buffer). Transformants were only obtained when 7 or 7.5 kV cm⁻¹ pulses were applied. Transformation efficiencies evaluated from the growth curves of transformed cells were between 10⁵ and 10⁷ transformants per microgram of plasmid DNA for five different replicon-based plasmids. Restriction nuclease digestion patterns of pJIR418 purified from transformed Clostridia and Escherichia coli were indistinguishable, indicating that heterologous DNA was structurally stable in the Clostridium strain. Copy numbers of 130, 70 and 10 were estimated from purification yield for pCTC1, pKNT19 and pJIR418, respectively. Journal of Industrial Microbiology & Biotechnology (2001) 27, 271–274. Received 12 September 2000/ Accepted in revised form 25 November 2000     

A comparison of the illness beliefs of people with angina and their peers: a questionnaire study

Background  

What people believe about their illness may affect how they cope with it. It has been suggested that such beliefs stem from those commonly held within society . This study compared the beliefs held by people with angina, regarding causation and coping in angina, with the beliefs of their friends who do not suffer from angina.     

seq++: analyzing biological sequences with a range of Markov-related models

SUMMARY: The seq++ package offers a reference set of programs and an extensible library to biologists and developers working on sequence statistics. Its generality arises from the ability to handle sequences described with any alphabet (nucleotides, amino acids, codons and others). seq++ enables sequence modelling with various types of Markov models, including variable length Markov models and the newly developed parsimonious Markov models, all of them potentially phased. Simulation modules are supplied for Monte Carlo methods. Hence, this toolbox allows the study of any biological process which can be described by a series of states taken from a finite set.     

Hyaluronan-mediated CD44 interaction with RhoGEF and Rho kinase promotes Grb2-associated binder-1 phosphorylation and phosphatidylinositol 3-kinase signaling leading to cytokine (macrophage-colony stimulating factor) production and breast tumor progression

In this study we have examined CD44 (a hyaluronan (HA) receptor) interaction with a RhoA-specific guanine nucleotide exchange factor (p115RhoGEF) in human metastatic breast tumor cells (MDA-MB-231 cell line). Immunoprecipitation and immunoblot analyses indicate that both CD44 and p115RhoGEF are expressed in MDA-MB-231 cells and that these two proteins are physically associated as a complex in vivo. The binding of HA to MDA-MB-231 cells stimulates p115RhoGEF-mediated RhoA signaling and Rho kinase (ROK) activity, which, in turn, increases serine/threonine phosphorylation of the adaptor protein, Gab-1 (Grb2-associated binder-1). Phosphorylated Gab-1 promotes PI 3-kinase recruitment to CD44v3. Subsequently, PI 3-kinase is activated (in particular, alpha, beta, gamma forms but not the delta form of the p110 catalytic subunit), AKT signaling occurs, the cytokine (macrophage-colony stimulating factor (M-CSF)) is produced, and tumor cell-specific phenotypes (e.g. tumor cell growth, survival and invasion) are up-regulated. Our results also demonstrate that HA/CD44-mediated oncogenic events (e.g. AKT activation, M-CSF production and breast tumor cell-specific phenotypes) can be effectively blocked by a PI 3-kinase inhibitor (LY294002). Finally, we have found that overexpression of a dominant-negative form of ROK (by transfection of MBA-MD-231 cells with the Rho-binding domain cDNA of ROK) not only inhibits HA/CD44-mediated RhoA-ROK activation and Gab-1 phosphorylation but also down-regulates oncogenic signaling events (e.g. Gab-1.PI 3-kinase-CD44v3 association, PI 3-kinase-mediated AKT activation, and M-CSF production) and tumor cell behaviors (e.g. cell growth, survival, and invasion). Taken together, these findings strongly suggest that CD44 interaction with p115RhoGEF and ROK plays a pivotal role in promoting Gab-1 phosphorylation leading to Gab-1.PI 3-kinase membrane localization, AKT signaling, and cytokine (M-CSF) production during HA-mediated breast cancer progression.     

Hyaluronan promotes signaling interaction between CD44 and the transforming growth factor beta receptor I in metastatic breast tumor cells

In this study we have examined the interaction between CD44 (a hyaluronan (HA) receptor) and the transforming growth factor beta (TGF-beta) receptors (a family of serine/threonine kinase membrane receptors) in human metastatic breast tumor cells (MDA-MB-231 cell line). Immunological data indicate that both CD44 and TGF-beta receptors are expressed in MDA-MB-231 cells and that CD44 is physically linked to the TGF-beta receptor I (TGF-betaRI) (and to a lesser extent to the TGF-beta receptor II (TGF-betaRII)) as a complex in vivo. Scatchard plot analyses and in vitro binding experiments show that the cytoplasmic domain of CD44 binds to TGF-betaRI at a single site with high affinity (an apparent dissociation constant (K(d)) of approximately 1.78 nm). These findings indicate that TGF-betaRI contains a CD44-binding site. Furthermore, we have found that the binding of HA to CD44 in MDA-MB-231 cells stimulates TGF-betaRI serine/threonine kinase activity which, in turn, increases Smad2/Smad3 phosphorylation and parathyroid hormone-related protein (PTH-rP) production (well known downstream effector functions of TGF-beta signaling). Most importantly, TGF-betaRI kinase activated by HA phosphorylates CD44, which enhances its binding interaction with the cytoskeletal protein, ankyrin, leading to HA-mediated breast tumor cell migration. Overexpression of TGF-betaRI by transfection of MDA-MB-231 cells with TGF-betaRIcDNA stimulates formation of the CD44.TGF-betaRI complex, the association of ankyrin with membranes, and HA-dependent/CD44-specific breast tumor migration. Taken together, these findings strongly suggest that CD44 interaction with the TGF-betaRI kinase promotes activation of multiple signaling pathways required for ankyrin-membrane interaction, tumor cell migration, and important oncogenic events (e.g. Smad2/Smad3 phosphorylation and PTH-rP production) during HA and TGF-beta-mediated metastatic breast tumor progression.     

Fluorescent derivatives of the GFP chromophore give a new insight into the GFP fluorescence process

The photophysical properties of synthetic compounds derived from the imidazolidinone chromophore of the green fluorescent protein were determined. Various electron-withdrawing or electron-donating substituents were introduced to mimic the effect of the chromophore surroundings in the protein. The absorption and emission spectra as well as the fluorescence quantum yields in dioxane and glycerol were shown to be highly dependent on the electronic properties of the substituents. We propose a kinetic scheme that takes into account the temperature-dependent twisting of the excited molecule. If the activation energy is low, the molecule most often undergoes an excited-state intramolecular twisting that leads it to the ground state through an avoided crossing between the S(1) and S(0) energy surfaces. For a high activation energy, the torsional motion within the compounds is limited and the ground-state recovery will occur preferentially by fluorescence emission. The excellent correlation between the fluorescence quantum yields and the calculated activation energies to torsion points to the above-mentioned avoided crossing as the main nonradiative deactivation channel in these compounds. Finally, our results are discussed with regard to the chromophore in green fluorescent protein and some of its mutants.     

CD44 interaction with ankyrin and IP3 receptor in lipid rafts promotes hyaluronan-mediated Ca2+ signaling leading to nitric oxide production and endothelial cell adhesion and proliferation

In this study, we have showed that aortic endothelial cells (GM7372A cell line) express CD44v10 [a hyaluronan (HA) receptor], which is significantly enriched in cholesterol-containing lipid rafts (characterized as caveolin-rich plasma membrane microdomains). HA binding to CD44v10 promotes recruitment of the cytoskeletal protein, ankyrin and inositol 1,4,5-triphosphate (IP3) receptor into cholesterol-containing lipid rafts. The ankyrin repeat domain (ARD) of ankyrin is responsible for binding IP3 receptor to CD44v10 at lipid rafts and subsequently triggering HA/CD44v10-mediated intracellular calcium (Ca2+) mobilization leading to a variety of endothelial cell functions such as nitric oxide (NO) production, cell adhesion and proliferation. Further analyses indicate (i) disruption of lipid rafts by depleting cholesterol from the membranes of GM7372A cells (using methyl-beta-cyclodextrin treatment) or (ii) interference of endogenous ankyrin binding to CD44 and IP3 receptor using overexpression of ARD fragments (by transfecting cells with ARDcDNA) not only abolishes ankyrin/IP3 receptor accumulation into CD44v10/cholesterol-containing lipid rafts, but also blocks HA-mediated Ca2+ signaling and endothelial cell functions. Taken together, our findings suggest that CD44v10 interaction with ankyrin and IP3 receptor in cholesterol-containing lipid rafts plays an important role in regulating HA-mediated Ca2+ signaling and endothelial cell functions such as NO production, cell adhesion and proliferation.