The neutralization of acidic coal mine lakes by additions of natural organic matter: a mesocosm test

Cylindrical polyethylene enclosures 3 m in length and 1 m in diameter reaching from the surface to the bottom were constructed in an acid (pH=3.1) lake on a coal surface mine in southern Illinois. Wheat straw was added to the enclosures to test the effects of dissimilatory sulfate reduction on water chemistry. Added straw increased sulfide concentrations, raised pH to 6.5, reduced O₂ and increased acid neutralizing capacity of the enclosed water columns when compared with a control enclosure and with the open lake. Generation of acid neutralizing capacity exceeded the standing stock of sulfide indicating that sulfide was removed either by precipitation of FeS or outgassing of H₂S. The pH and acid neutralizing capacity within the enclosures eventually returned to the level of the surrounding lake because of water exchange around the enclosure walls. Our results show that additions of organic matter to acid surface mine lakes result in the generation of acid neutralizing capacity.     

Microsecond Dynamics During the Binding-induced Folding of an Intrinsically Disordered Protein

Tau is an intrinsically disordered protein implicated in many neurodegenerative diseases. The repeat domain fragment of tau, tau-K18, is known to undergo a disorder to order transition in the presence of lipid micelles and vesicles, in which helices form in each of the repeat domains. Here, the mechanism of helical structure formation, induced by a phospholipid mimetic, sodium dodecyl sulfate (SDS) at sub-micellar concentrations, has been studied using multiple biophysical probes. A study of the conformational dynamics of the disordered state, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS) has indicated the presence of an intermediate state, I, in equilibrium with the unfolded state, U. The cooperative binding of the ligand (L), SDS, to I has been shown to induce the formation of a compact, helical intermediate (IL₅) within the dead time (∼37 µs) of a continuous flow mixer. Quantitative analysis of the PET-FCS data and the ensemble microsecond kinetic data, suggests that the mechanism of induction of helical structure can be described by a U ↔ I ↔ IL₅ ↔ FL₅ mechanism, in which the final helical state, FL₅, forms from IL₅ with a time constant of 50–200 µs. Finally, it has been shown that the helical conformation is an aggregation-competent state that can directly form amyloid fibrils.     

Activation of the PDGF β Receptor by a Persistent Artificial Signal Peptide

Most eukaryotic transmembrane and secreted proteins contain N-terminal signal peptides that mediate insertion of the nascent translation products into the membrane of the endoplasmic reticulum. After membrane insertion, signal peptides typically are cleaved from the mature protein and degraded. Here, we tested whether a small hydrophobic protein selected for growth promoting activity in mammalian cells retained transforming activity while also acting as a signal peptide. We replaced the signal peptide of the PDGF β receptor (PDGFβR) with a previously described 29-residue artificial transmembrane protein named 9C3 that can activate the PDGFβR in trans. We showed that a modified version of 9C3 at the N-terminus of the PDGFβR can function as a signal peptide, as assessed by its ability to support high level expression, glycosylation, and cell surface localization of the PDGFβR. The 9C3 signal peptide retains its ability to interact with the transmembrane domain of the PDGFβR and cause receptor activation and cell proliferation. Cleavage of the 9C3 signal peptide from the mature receptor is not required for these activities. However, signal peptide cleavage does occur in some molecules, and the cleaved signal peptide can persist in cells and activate a co-expressed PDGFβR in trans. Our finding that a hydrophobic sequence can display signal peptide and transforming activity suggest that some naturally occurring signal peptides may also display additional biological activities by interacting with the transmembrane domains of target proteins.     

In vitro culture of human chondrocytes (1): A novel enhancement action of ferrous sulfate on the differentiation of human chondrocytes

Chondrogenic differentiation of mesenchymal cells is generally thought to be initiated by the inductive action of specific growth factors and depends on intimate cell-cell interactions. The aim of our investigation was to characterize the influences of basic fibroblast growth factor (bFGF) and ferroussulfate (FeSO₄) on proliferation and differentiation of human articular chondrocytes (HAC). This is the first report of the effects of FeSO₄ on chondrogenesis of HAC. Multiplied chondrocytes of hip and shoulder joints were cultured in chondrocyte growth medium supplemented with bFGF, FeSO₄, or both bFGF + FeSO₄ for4weeks. A 20 μl aliquot of a cell suspension containing2 × 10⁷ cells ml⁻¹ was delivered onto each well of 24-well tissue culture plates. Cells cultured with the growth medium only was used as a control. Alamar blue and alcian blue staining were done to determine the chondrocyte proliferation and differentiation, respectively, after 4 weeks. The samples exposed to bFGF, FeSO₄, and combination of both indicated sufficient cell proliferation similar to the control level. Differentiations of the HAC exposed to bFGF, FeSO₄,and bFGF + FeSO₄ were 1.2-, 2.0-, and 2.2-fold of the control, respectively. Therefore, chondrocyte differentiation was significantly enhanced by the addition of FeSO₄ andbFGF + FeSO₄. The combined effects of bFGF and FeSO₄ were additive, rather than synergistic. These results suggest that treatment with ferrous sulfate alone or in combination with basic fibroblast growth factor etc, is a powerful tool to promote the differentiation of HAC for the clinical application. This revised version was published online in August 2006 with corrections to the Cover Date.     

Monoclonal antibodies against beta-galactoside-binding lectin of chick embryo recognizes conformational change of the antigen

Monoclonal antibodies against chick embryonic beta-galactoside-binding lectin were obtained. One of the monoclonal antibodies was ineffective in Western blotting and seemed to be unable to bind the SDS-denatured lectin. When the native lectin was dotted on a nitrocellulose filter and subjected to denaturation by treatment with SDS, urea or heat, binding of this antibody no longer occurred, though other monoclonal antibodies bound normally. This antibody seems to have been raised against an epitope which is destroyed upon denaturation.     

Syntheses of novel azasugar-containing mimics of heparan sulfate fragments as potential heparanase inhibitors

A series of eight novel, highly modified mono-, di- and trisaccharide derivatives, each containing an iminoalditol moiety, has been synthesized in the quest for potential heparanase inhibitors.     

Proteoglycan from bovine follicular fluid enhances an acrosome reaction in bovine spermatozoa

Bovine epididymal and ejaculated spermatozoa were incubated for 22 and 9.5 h respectively, in a chemically defined medium. The percentages of sperm exhibiting an acrosome reaction were determined morphologically after fixing and staining specimens. Addition of bovine follicular proteoglycan or chondroitin sulfates ABC significantly increased the incidence of acrosome reaction. The stimulatory effects of the proteoglycan or chondroitin sulfates were negated by exposure to the enzyme chondroitinase ABC. Viability of spermatozoa was not affected by the various experimental treatments. Transmission electron microscopy of spermatozoa showed that vesiculation had occurred between the plasma and outer acrosomal membrane. These results suggest that proteoglycan present in follicular fluid at the time of ovulation may promote the acrosome reaction which precedes the ability of sperm to fertilize an ovum.     

Activation of pigeon erythrocyte adenylate cyclase by cholera toxin partial purification of an essential macromolecular factor from horse erythrocyte cytosol

A cytosolic, macromolecular factor required for the cholera toxin-dependent activation of pigeon erythrocyte adenylate cyclase and cholera toxin-dependent ADP-ribosylation of a membrane-bound 43 000 dalton polypeptide has been purified 1100-fold from horse erythrocyte cytosol using organic solvent precipitation and heat treatment. This factor, 13 000 daltons, does not absorb to anionic or cationic exchange resins, is sensitive to trypsin or 10% trichloroacetic acid and is not extractable by diethyl ether. Activation of adenylate cyclase by cholera toxin requires the simultaneous presence of ATP (including possible trace GTP), NAD⁺, dithiothreitol, cholera toxin, membranes and the cytosolic macromolecular factor. Reversal of cholera toxin activation of adenylate cyclase, and of the toxin-dependent ADP-ribosylation, requires the presence of the cytosolic factor. The ability of the purified cytosolic factor to influence the hormonal sensitivity of liver membrane adenylate cyclase may provide clues to its physiological functions.