Analysis of state-specific phosphorylation of proteins by two-dimensional gel electrophoresis approach

In this paper we focus on the detection of specific state of protein phosphorylation within a complex protein mixture separated by two-dimensional gel electrophoresis followed by immunoblotting. The availability of antibodies that specifically recognize the phosphorylated residue(s) of proteins make this approach feasible as exemplified by the study of the regulatory mechanisms of the cell cycle. The major advantage of the presented approach is its relative simplicity and sensitivity that allows specific detection of protein phosphorylation and distinguishes different phosphorylation sites of target protein. Current findings demonstrate that this method represents a reasonable alternative to the use of other tools to study protein phosphorylation.     

Galactose-substituted alginate: preliminary characterization and study of gelling properties

Coupling of alginate with 1-amino-1-deoxygalactose in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide results in a substituted polymer containing galactose side linked via an amide bond. To clarify the degree and pattern of substitution, a (1)H NMR study on the anomeric region of modified alginate, polymannuronate, alginate enriched in guluronic acid (G-enriched alginate), and polyalternating MG, was carried out (G, alpha-l-guluronic acid; M, beta-d-mannuronic acid). From the resonance of the proton at position 1 of galactosylamine, it was possible to determine the amount of galactose linked to mannuronic and to guluronic residues, respectively. Furthermore, (1)H NMR spectroscopy revealed a higher reactivity of guluronic residues for low degrees of conversion. Modified alginates with 7% and 19% of substitution are both able to form stable beads in the presence of calcium ions. The effect of galactose substitution on the dimensions, swelling, and stability of the beads has been studied and the cytotoxicity of the modified polymer evaluated in preliminary biological tests.     

A-hydroxyphosphonate oligonucleotides: a promising DNA type?

The synthesis of monomers (S)-1, (R)-1 and 2 derived from (5'S)-, (5'R)-2'-deoxythymidine-5'-C-phosphonic acids and 2',5'-dideoxythymidine-5'-C-phosphonic acids was elaborated. The protection of the 5'-hydroxyl by the methoxycarbonyl group was a key step of the synthesis. Prepared monomers were used for the solid-phase assembly of several types oligothymidylate 15-mers (S)-3, (S)-4, (S)-5, (R)-4 and (R)-5 containing the chiral 3'-O-P-CH(OH)-5' internucleotide linkage. Their hybridization properties with dA15 and rA15 were studied as well as their resistance against nuclease cleavage.     

Quantitative proteomics of lymphocytes

Lymphocytes are the best-studied higher eukaryote cells. In this report, quantitative relationships of the protein components in resting cell, blast cell and plasma cell types are evaluated. The comparison of these cell types leads to the conclusion that resting cells synthesize about one-twentieth of the protein species as compared to blast cells. Blast cells seem to be metabolically the most robust lymphocyte type. Plasma cells are geared towards synthesis of one main product (antibody in B plasma cells), while most of the synthesis of other protein species (including those for housekeeping and repair) decreases as the messages decay. Although the data presented in this communication allow a meaningful comparison of three cell populations, they are far from providing a full picture. Both silver staining and radiofluorography depict only proteins of high or intermediate abundance. Silver staining misses most proteins present at <10 000 copies/cell, while radiofluorography misses all those proteins with slow turnover (and those with no methionine residue in their sequence). The detection of 1100 spots in the blast cell-related radiofluorograph includes visualization of some 97-99% of protein mass, but some 3900 polypeptide species in the remaining 1-3% of protein mass will pass undetected. This protein mass (0.7-2 pg) reflects some 2500-7500 copies of each of those 3900 polypeptide species that are present in the cell below the detection limit. The work emphasizes that full understanding of cellular function can be achieved only if quantitative aspects of cell inventory are considered.     

Different responses of tobacco antioxidant enzymes to light and chilling stress

The effect of elevated light treatment (25 degrees C, PPFD 360 mumol m-2 sec-1) or chilling temperatures combined with elevated light (5 degrees C, PPFD 360 mumol m-2 sec-1) on the activity of six antioxidant enzymes, guaiacol peroxidases, and glutathione peroxidase (GPx, EC 1.11.1.9) protein accumulation were studied in tobacco Nicotiana tabacum cv. Petit Havana SR1. Both treatments caused no photooxidative damage, but chilling caused a transient wilting. The light treatment increased the activities of ascorbate peroxidase (APx, EC 1.11.1.11) and guaiacol peroxidases while catalase (EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) were unchanged. In contrast, chilling treatment did not increase any of the antioxidant enzyme activities, but decreased catalase and to a lesser extent DHAR activities. Glutathione peroxidase protein levels increased sporadically under light treatment and constantly under chilling. Both chilling and light stress caused induction of glutathione synthesis and accumulation of oxidised glutathione, although the predominant part of the glutathione pool remained in the reduced form. Antioxidant enzymes from the chilling treated plants were measured at both 25 degrees C and 5 degrees C. Measurements at 5 degrees C revealed a 3-fold reduction in catalase activity, compared with that measured at 25 degrees C, indicating that the overall reduction in catalase after four days of chilling was approximately 10-fold. The overall reduction in activity for the other antioxidant enzymes after four days of chilling was 2-fold for GR and APx, 1.5-fold for MDHAR, 3.5-fold for DHAR. The activity of SOD was the same at 25 and 5 degrees C. These results indicate that catalase and DHAR are most strongly affected by the chilling treatment and may be the rate-limiting factor of the antioxidant system at low temperatures.     

On-line analysis of gap junctions reveals more efficient electrical than dye coupling between islet cells

Pancreatic beta-cells constitute a well-communicating multicellular network that permits a coordinated and synchronized signal transmission within the islet of Langerhans that is necessary for proper insulin release. Gap junctions are the molecular keys that mediate functional cellular connections, which are responsible for electrical and metabolic coupling in the majority of cell types. Although the role of gap junctions in beta-cell electrical coupling is well documented, metabolic communication is still a matter of discussion. Here, we have addressed this issue by use of a fluorescence recovery after photobleaching (FRAP) approach. This technique has been validated as a reliable and noninvasive approach to monitor functional gap junctions in real time. We show that control pancreatic islet cells did not exchange a gap junction-permeant molecule in either clustered cells or intact islets of Langerhans under conditions that allowed cell-to-cell exchange of current-carrying ions. Conversely, we have detected that the same probe was extensively transferred between islet cells of transgenic mice expressing connexin 32 (Cx32) that have enhanced junctional coupling properties. The results indicate that the electrical coupling of native islet cells is more extensive than dye communication. Dye-coupling domains in islet cells appear more restricted than previously inferred with other methods.     

Expression of truncated PrP targeted to Purkinje cells of PrP knockout mice causes Purkinje cell death and ataxia

PrP knockout mice with disruption of only the PrP-encoding region (Zürich I-type) remain healthy, whereas mice with deletions extending upstream of the PrP-encoding exon (Nagasaki-type) suffer Purkinje cell loss and ataxia, associated with ectopic expression of Doppel in brain, particularly in Purkinje cells. The phenotype is abrogated by co-expression of full-length PrP. Doppel is 25% similar to PrP, has the same globular fold, but lacks the flexible N-terminal tail. We now show that in Zürich I-type PrP-null mice, expression of N-terminally truncated PrP targeted to Purkinje cells also leads to Purkinje cell loss and ataxia, which are reversed by PrP. Doppel and truncated PrP probably cause Purkinje cell degeneration by the same mechanism.