Effects of phosphorylation of protein phosphatase 1 by pp60v-src on the interaction of the enzyme with substrates and inhibitor proteins

Phosphorylation of protein phosphatase 1 by pp60v-src decreased its activity towards phosphorylase kinase and glycogen synthase as well as towards phosphorylase a. Kinetic experiments indicated that the primary effect of phosphorylation was to increase the Km for each of the substrate proteins. There was little or no change in the Vmax for the reactions. The possibility that phosphorylation of protein phosphatase 1 altered its regulation by inhibitors-1 and -2 was also examined. Phosphorylation of protein phosphatase 1 did not prevent the reversible inhibition of the enzyme by inhibitor-1 or inhibitor-2 nor did it prevent the association of inhibitor-2 with protein phosphatase 1 to form the MgATP-dependent protein phosphatase. Protein phosphatase 1 is not a substrate for pp60v-src when it is complexed with inhibitor-2 to form the inactive MgATP-dependent protein phosphatase. Here we have shown that protein phosphatase 1 is also not phosphorylated by pp60v-src following activation of the MgATP-dependent protein phosphatase with glycogen synthase kinase-3 and MgATP. This indicates that the inability of pp60v-src to phosphorylate protein phosphatase 1 is not due to the change in protein phosphatase 1 conformation which accompanies the inactivation of the MgATP-dependent protein phosphatase. Rather, it appears to be the result of steric hindrance by inhibitor-2. This suggests that the pp60v-src phosphorylation site is closely associated with the inhibitor-2 binding site involved in the formation of the MgATP dependent protein phosphatase. The pp60v-src phosphorylation site was previously localized to a small (Mr less than or equal to 4000) domain which can be selectively degraded by chymotrypsin. Here we have shown that chymotryptic digestion increased the Km of unphosphorylated protein phosphatase 1 for each of the three phosphoprotein substrates used in this study. This effect was similar to that observed after phosphorylation of protein phosphatase 1. These results indicate that the pp60v-src phosphorylation site is in a region of protein phosphatase 1 which influences substrate binding and which may be near the active site.     

Gene marking of human neural stem/precursor cells using green fluorescent proteins

BACKGROUND: Ex vivo gene therapy and cell replacement in the nervous system may provide therapeutic opportunities for neurodegenerative disorders. The development of optimal gene marking procedures for human neural stem cells (hNSCs) is crucial for the success of these strategies, in order to provide a correct understanding of the biology of transplanted cells. METHODS: hNSCs were modified to express various members of the green fluorescent protein family of proteins. Both DNA and retroviral expression vectors were used. Cells were analyzed for transgene expression under transient and stable expression schemes, and in the presence or absence of drug selection, by fluorescence microscopy, histochemistry, immunocytochemistry, immunoblotting, RT-PCR and flow cytometry. Genetically marked cells were analyzed in vivo after intrastriatal transplantation in neonatal rats. RESULTS: Using the same experimental procedures, we have compared Aequorea victoria enhanced green fluorescent protein (Av-eGFP) and Renilla raniformis GFP (Rh-GFP, h- from humanized) for the purpose of gene marking of hNSCs. Our findings revealed practical problems for the derivation of stable Av-eGFP-expressing hNSCs, whereas Rh-GFP could be well expressed. In a second phase of the study, stable Rh-GFP-expressing clonal hNSCs were derived. Rh-GFP did not interfere with the differentiation potential of the cells, and expression levels were identical between division and differentiation conditions. Thirdly, in vivo, we have confirmed the usefulness of Rh-GFP for the study of the transplant performance of hNSCs, and demonstrated that Rh-GFP does not interfere with multipotency and differentiation. CONCLUSIONS: Searching for suitable and useful reporter genes, we have found that Rh-GFP works efficiently for the purpose of stable gene marking of hNSCs, and is highly useful in vivo. The nature, properties, and possible side effects of marker genes are discussed, since these are important parameters to consider in gene marking studies involving hNSCs.     

Oxidative stress and the use of antioxidants in diabetes: Linking basic science to clinical practice

Cardiovascular complications, characterized by endothelial dysfunction and accelerated atherosclerosis, are the leading cause of morbidity and mortality associated with diabetes. There is growing evidence that excess generation of highly reactive free radicals, largely due to hyperglycemia, causes oxidative stress, which further exacerbates the development and progression of diabetes and its complications. Overproduction and/or insufficient removal of these free radicals result in vascular dysfunction, damage to cellular proteins, membrane lipids and nucleic acids. Despite overwhelming evidence on the damaging consequences of oxidative stress and its role in experimental diabetes, large scale clinical trials with classic antioxidants failed to demonstrate any benefit for diabetic patients. As our understanding of the mechanisms of free radical generation evolves, it is becoming clear that rather than merely scavenging reactive radicals, a more comprehensive approach aimed at preventing the generation of these reactive species as well as scavenging may prove more beneficial. Therefore, new strategies with classic as well as new antioxidants should be implemented in the treatment of diabetes.     

Isolation and taxonomic affiliation of N-heterocyclic aromatic hydrocarbon-transforming bacteria

The azaarenes (nitrogen-containing heterocyclic aromatic hydrocarbons) are products of incomplete combustion processes and thus are widely distributed with tar and oil products in the environment. Despite their adverse organoleptic, toxic, and carcinogenic characteristics, the biodegradability and fate of multi-ring azaarenes have received little attention. This work demonstrates the presence of genetically diverse azaarene-degrading bacteria in coal tar-contaminated soils. Thirty-eight bacterial strains able to transform the three-ring azaarenes, 5,6- and 7,8-benzoquinoline, phenanthridine, phenazine, or acridine, were isolated. Only seven of these strains grew in liquid medium on the specific azaarene compounds on which they were isolated using plates; and the rest transformed the azaarenes without growth. Taxonomic characterization by 16S ribosomal DNA sequencing revealed that our enrichment technique provided a diversity of 18 different azaarene-transforming bacterial species. Only a few strains were able to mineralize the homocyclic analogue, phenanthrene. Several of the isolates, e.g., Dyadobacter fermentans, Methylopila capsulata, and Agrobacterium tumefaciens, were related to genera relatively unknown with respect to the biodegradation of xenobiotic compounds. These strains can provide further information on the fate of azaarenes in the environment.     

Recent glimpses of the elusive spindle matrix

A stationary spindle matrix has been proposed on theoretical grounds to help mediate force production at the mitotic spindle. Direct molecular evidence for the existence of such a matrix has the potential to profoundly influence our view of the molecular mechanisms leading to chromosome segregation during mitosis. Three recent papers suggest that the spindle matrix may be more than a theoretical idea.     

Proton magic angle spinning NMR reveals new features in photodynamically treated bacteria

The bacterium Propionibacterium acnes is light-sensitive due to porphyrin-induced photosensitization. The light sensitivity increases with incubation of 5-aminolevulinic acid, ALA. For the first time, 1H magic angle spinning NMR spectroscopy is used to describe the photoinduced changes in the bacterium after ALA incubation. Successful photosensitization was performed with light-emitting diodes in the blue and red regions (430 and 654 nm, respectively). The irradiation setup, suitable for irradiation of bacterium suspensions in petri dishes is described. For NMR studies blue light diodes with about 90 micromol/m2s were chosen. After blue light irradiation, the endogenous glycine betaine, proline, glutamate and choline levels in P. acnes decreased with increasing irradiation time. For sublethal light doses (50% survival fraction), the endogenous glycine betaine level decreased 80% on average. The corresponding percentages for proline, choline and glutamate were about 40, 25 and 10, respectively. It is hypothesized that the irradiation, inducing porphyrin photosensitization amplified by ALA incubation, leads to elimination of the osmolyte glycine betaine and possibly also proline by so-called regulatory volume decrease (RVD) mechanisms. These mechanisms are known to be active in several prokaryotic and eukaryotic cells when exposed to hypotonic stress. They are also known to be present in several eukaryotic cells during photodynamic therapy (PDT) exposure leading to hypotonoc stress. The findings contribute to the knowledge of the inactivation mechanisms of P. acnes in photosensitization, and could therefore be of interest in the efforts to use PDT as treatment of the acne disease.