Qualitative and quantitative observations on the structure of the Schwann cells in myelinated fibres

Various morphological features of the Schwann cells of myelinated fibres in the lizard thoracic spinal roots were studied, and, when possible, quantified using morphometric methods. About 0.8% of the Schwann cells are binucleate and some display clusters of microvilli along the internodes. The percentages of the cytoplasmic area of the Schwann cell occupied by the following cytoplasmic components were determined: mitochondria, Golgi apparatus, granular endoplasmic reticulum, smooth endoplasmic reticulum, multivesicular bodies, dense bodies, autophagic vacuoles, peroxisome-like bodies, lipofuscin granules and lipid droplets. Linear relationships were found between the sectional areas of the mitochondria and granular endoplasmic reticulum of the Schwann cell and both the length of the profile of the Schwann cell plasma membrane and the size of the related axon. The results obtained are compatible both with the hypothesis that the mitochondria and granular endoplasmic reticulum of the Schwann cell are involved in the production and storage of proteins for the plasma membrane of this cell, and with the hypothesis that these organelles are involved in the production and storage of protein metabolites which are subsequently transferred to the related axons.     

Mesoscopic gel at low agarose concentration in water: a dynamic light scattering study.

Previous work in our laboratory has shown that at very low agarose concentration in water gelation still occurs within mutually disconnected, high concentration regions generated by spinodal demixing. The freely diffusing particles obtained in these conditions are studied in the present work by depolarized dynamic light scattering and probe diffusion experiments. These particles are found to behave as large (in fact, mesoscopic) polymer fibers entangled in a continuously rearranged mesh with scaling parameters typical of partially flexible, neutral chains. The present results allow specifying the notion of mesoscopic gelation. They also reveal that the same symmetry-breaking mechanism that allows macroscopic gelation at polymer concentrations well below the threshold for random cross-link percolation generates additional and unexpected phenomena.     

Continuous culture system for production of biopolymer levan using erwinia herbicola

The optimal production of the fructan biopolymer levan by the bacterium Erwinia herbicola was investigated, including variations in nitrogen, carbon and phosphorous sources, pH, incubation time, culture yields up to 19% by weight produced based on conversion of sucrose as the carbon source when grown in a continuous culture system and processed by tangential flow filtration. Product identity was confirmed with gas chromatography (GC) and (13)C nuclear magnetic resonance (NMR). Gel permeation chromatography (GPC) and low-angle laser light scattering (LALLS) determination of the molecular weight of the product showed a significant difference in molecular weight values dependent on the method of analysis. Analysis by GPC resulted in molecular weight one order of magnitude lower than LALLS independent of sample, underscoring the unusual nature of this biopolymer.     

Temperature dependence of the effects of some monohydric alcohols on the oxygen affinity of hemoglobin: Determination and analysis of thermodynamic parameters

We studied the effects of methanol, ethanol, iso-propanol, and n-propanol on the reaction of hemoglobin with oxygen at various temperatures. The analysis of the results in terms of the Monod-Wyman-Changeux model allowed determination of the overall contribution of the alcohols to the standard enthalpy and entropy differences between the T and R states of hemoglobin. A phenomenological approach allowed us to obtain separately the contributions related to the variations of the bulk dielectric constant of the solvent (bulk electrostatic contributions) and the contributions related to other effects (non-bulk-electrostatic contributions). The values of non-bulk-electrostatic contributions to ΔΔH and ΔΔS supported the suggestion that these contributions are mainly related to protein-solvent hydrophobic interactions.     

Biochemical characterization of a CDC6-like protein from the crenarchaeon Sulfolobus solfataricus

Cdc6 proteins play an essential role in the initiation of chromosomal DNA replication in Eukarya. Genes coding for putative homologs of Cdc6 have been also identified in the genomic sequence of Archaea, but the properties of the corresponding proteins have been poorly investigated so far. Herein, we report the biochemical characterization of one of the three putative Cdc6-like factors from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (SsoCdc6-1). SsoCdc6-1 was overproduced in Escherichia coli as a His-tagged protein and purified to homogeneity. Gel filtration and glycerol gradient ultracentrifugation experiments indicated that this protein behaves as a monomer in solution (molecular mass of about 45 kDa). We demonstrated that SsoCdc6-1 binds single- and double-stranded DNA molecules by electrophoretic mobility shift assays. SsoCdc6-1 undergoes autophosphorylation in vitro and possesses a weak ATPase activity, whereas the protein with a mutation in the Walker A motif (Lys-59 --> Ala) is completely unable to hydrolyze ATP and does not autophosphorylate. We found that SsoCdc6-1 strongly inhibits the ATPase and DNA helicase activity of the S. solfataricus MCM protein. These findings provide the first in vitro biochemical evidence of a functional interaction between a MCM complex and a Cdc6 factor and have important implications for the understanding of the Cdc6 biological function.     

Purification and characterization of recombinant spider silk expressed in Escherichia coli

A partial cDNA clone, from the 3′ end of the dragline silk gene was isolated from Nephila clavipes major ampullate glands. This clone contains a 1.7-kb insert, consisting of a repetitive coding region of 1.4-kb and a 0.3-kb nonrepetitive coding region; 1.5-kb of the 1.7-kb fragment was cloned into Escherichia coli and a␣43-kDa recombinant silk protein was expressed. Characterization of the purified protein by Western blot, amino acid composition analysis, and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry confirms it to be spider dragline silk. Received: 7 April 1997 / Received revision: 24 July 1997 / Accepted: 25 August 1997     

Irreversible gelation of thermally unfolded proteins: structural and mechanical properties of lysozyme aggregates

The formation of protein aggregates is important in many fields of life science and technology. The morphological and mechanical properties of protein solutions depend upon the molecular conformation and thermodynamic and environmental conditions. Non-native or unfolded proteins may be kinetically trapped into irreversible aggregates and undergo precipitation or gelation. Here, we study the thermal aggregation of lysozyme in neutral solutions. We characterise the irreversible unfolding of lysozyme by differential scanning calorimetry. The structural properties of aggregates and their mechanisms of formation with the eventual gelation are studied at high temperature by spectroscopic, rheological and scattering techniques. The experiments show that irreversible micron-sized aggregates are organised into larger clusters according to a classical mechanism of diffusion and coagulation, which leads to a percolative transition at high concentrations. At a smaller length scale, optical and atomic force microscopy images reveal the existence of compact aggregates, which are the origin of the aggregation irreversibility.     

Modification of the detrimental effect of TNF‐α on human endothelial progenitor cells by fasudil and Y27632

Exposure of human endothelial progenitor cells (EPCs) to tumor necrosis factor‐α (TNF‐α) reduced their number and biological activity. Yet, signal transduction events linked to TNF‐α action are still poorly understood. To address this issue, we examined the possible effect of fasudil and Y27632, two inhibitors of Rho kinase pathway, which is involved in endothelial dysfunction, atherosclerosis, and in‐ flammation. Results demonstrated that incubation with fasudil starting from 50 μM but not Y27632 determined a dose‐dependent improvement of EPC number during exposure to TNF‐α (P < 0.05 vs. TNF‐α alone). Analysis of the signal transduction pathway activated by TNF‐α revealed that the increased expression of p‐p38 was not significantly altered by fasudil. Instead, fasudil blocked the TNF‐α induced phosphorylation of Erk1/2 (P < 0.05 vs. TNF‐α) as well as the inhibitor of Erk1/2‐specific phosphorylated form, i.e., PD98059 (P < 0.05 vs. TNF‐α). These results were confirmed by analysis of these kinases by confocal microscopy. Finally, 2D‐DIGE and MALDI‐TOF/TOF analysis of EPCs treated with fasudil revealed increased expression levels of an actin‐related protein and an adenylyl cyclase associated protein and decreased expression levels of proteins related to radical scavenger and nucleotide metabolism. These findings suggest that fasudil positively affects EPC number and that other major signals might take part to this complex pathway. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:351–360, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20345