Helmut W. Klein

Transplantation of bone marrow-derived mesenchymal stem cells rescue photoreceptor cells in the dystrophic retina of the rhodopsin knockout mouse

BackgroundRetinitis pigmentosa belongs to a large group of degenerative diseases of the retina with a hereditary background. It involves loss of retinal photoreceptor cells and consequently peripheral vision. At present there are no satisfactory therapeutic options for this disease. Just recently the use of mesenchymal stem cells has been discussed as one therapeutical option for retinal degeneration, as they have been shown to differentiate into various cell types, including photoreceptor cells. In this article we wanted to investigate the potency of mesenchymal stem cells to induce rescue effects in an animal model for retinitis pigmentosa, the rhodopsin knockout mouse.MethodsFor the experiments, three experimental groups of 10 animals each were formed. The first group consisted of untreated rhodopsin knockout (rho-/-) animals used as controls. The second group consisted of rho-/- mice that had received an injection of mouse mesenchymal stem cells, which were transduced using an adenoviral vector containing the sequence for the green fluorescent protein (GFP) prior to transplantation. In the third sham group, animals received an injection of medium only. Thirty-five days after transplantation, GFP-expressing cells were detected in whole-mount preparations of the retinas as well as in cryostat sections. For the detection of rescue effects, semi-thin sections of eyes derived from all experimental groups were produced. Furthermore, rescue effects were also analysed ultrastructurally in ultrathin sections.ResultsHistological analysis revealed that after transplantation, cells morphologically integrated not only into the retinal pigment epithelium but also into layers of the neuroretina displaying neuronal and glial morphologies. Furthermore, significant rescue effects, as demonstrated by the occurrence of preserved photoreceptor cells, were detected.ConclusionsOur data indicate that mesenchymal stem cells can prolong photoreceptor survival in the rhodopsin knockout mouse, also providing evidence of a therapeutical benefit in retinitis pigmentosa.

Identification of Ser-1275 and Ser-1309 as autophosphorylation sites of the insulin receptor.

We have identified Ser‐1275 and Ser‐1309 as novel serine autophosphorylation sites by direct sequencing of HPLC‐purified tryptic phosphopeptides of the histidine‐tagged insulin receptor kinase IRKD‐HIS. The corresponding peptides (Ser‐1275, amino acids 1272–1292; Ser‐1309, amino acids 1305–1313) have been detected in the HPLC profiles of both the soluble kinase IRKD, which contains the entire cytoplasmic domain of the insulin receptor β‐subunit, and the insulin receptor purified from human placenta. In contrast, a kinase negative mutant, IRKD‐K1018A, did not undergo phosphorylation at either the tyrosine or serine residues, strongly suggesting that insulin receptor kinase has an intrinsic activity to autophosphorylate serine residues.

X-ray structure of glutathione S-transferase from Schistosoma japonicum in a new crystal form reveals flexibility of the substrate-binding site

The crystal structure of the 26 kDa glutathione S-transferase from Schistosoma japonicum (SjGST) was determined at 3 A resolution in the new space group P2(1)2(1)2(1). The structure of orthorhombic SjGST reveals unique features of the ligand-binding site and dimer interface when compared with previously reported structures. SjGST is recognized as the major detoxification enzyme of S. japonicum, a pathogenic helminth causing schistosomiasis. As resistance against the established inhibitor of SjGST, praziquantel, has been reported these results might prove to be valuable for the development of novel drugs.

Autophosphorylation of the two C-terminal tyrosine residues Tyr1316 and Tyr1322 modulates the activity of the insulin receptor kinase in vitro.

Previously, several studies have demonstrated that autophosphorylation of the C‐terminal tyrosine residues (Tyr1316 and Tyr1322) affects the signaling properties of the insulin receptor in vivo. To assess the biochemical consequences of the C‐terminal phosphorylation in vitro, we have constructed, purified and characterized 45 kDa soluble insulin receptor kinase domains (IRKD), either with (IRKD) or without (IRKD‐Y2F) the two C‐terminal tyrosine phosphorylation sites, respectively. According to HPLC phosphopeptide mapping, autophosphorylation of the three tyrosines in the activation loop of the IRKD‐Y2F kinase (Tyr1146, Tyr1150, and Tyr1151) was not affected by the mutation. In addition, the Y2F mutation did not significantly change the K m values for exogenous substrates. However, the mutation in IRKD‐Y2F resulted in a decrease in the maximum velocities of the phosphotransferase reaction in substrate phosphorylation reactions. Moreover, the exchange of the tyrosines in IRKD‐Y2F led to an increase in the apparent K m values for ATP, suggesting a cross‐talk of the C‐terminus and the catalytic domain of the enzyme. In addition, as judged by size exclusion chromatography, conformational changes of the enzyme following autophosphorylation were abolished by the removal of the two C‐terminal tyrosines. These data suggest a regulatory role of the two C‐terminal phosphorylation sites in the phosphotransferase activity of the insulin receptor.

14-3-3 binding to the IGF-1 receptor is mediated by serine autophosphorylation1

The phosphoserine‐binding 14‐3‐3 proteins have been implicated in playing a role in mitogenic and apoptotic signaling pathways. Binding of 14‐3‐3 proteins to phosphoserine residues in the C‐terminus of the insulin‐like growth factor‐1 receptor (IGF‐1R) has been described to occur in a variety of cell systems, but the kinase responsible for this serine phosphorylation has not been identified yet. Here we present evidence that the isolated dimeric insulin‐like growth factor‐1 receptor kinase domain (IGFKD) contains a dual specific (i.e. tyrosine/serine) kinase activity that mediates autophosphorylation of C‐terminal serine residues in the enzyme. From the total phosphate incorporation of ∼4 mol per mol kinase subunit, 1 mol accounts for serine phosphate. However, tyrosine autophosphorylation proceeds more rapidly than autophosphorylation of serine residues (t 1/2∼1 min vs. t 1/2∼5 min). Moreover, dot‐blot and far‐Western analyses reveal that serine autophosphorylation of IGFKD is sufficient to promote binding of 14‐3‐3 proteins in vitro. The proof that dual kinase activity of IGFKD is necessary and sufficient for 14‐3‐3 binding was obtained with an inactive kinase mutant that was phosphorylated on serine residues in a stoichiometric reaction with the catalytically active enzyme. Thus, the IGF‐1R itself might be responsible for the serine autophosphorylation which leads to recognition of 14‐3‐3 proteins in vivo.