[18F]FEAC and [18F]FEDAC: Two novel positron emission tomography ligands for peripheral-type benzodiazepine receptor in the brain

[¹⁸F]FEAC ([¹⁸F]4a) and [¹⁸F]FEDAC ([¹⁸F]4b) were developed as two novel positron emission tomography (PET) ligands for peripheral-type benzodiazepine receptor (PBR). [¹⁸F]4a and [¹⁸F]4b were synthesized by fluoroethylation of precursors 8a and 8b with [¹⁸F]FCH₂CH₂Br ([¹⁸F]9), respectively. Small-animal PET scan for a neuroinflammatory rat model showed that the two radioligands had high uptakes of radioactivity in the kainic acid-infused striatum, a brain region where PBR density was increased.     

Inhibition of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase by ß-D-4-O-sulfo-N-acetylgalactosaminides bearing various hydrophobic aglycons

N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate to position 6 of GalNAc(4SO₄) residues of chondroitin sulfate to yield chondroitin sulfate E (CS-E). We have previously demonstrated that phenyl-ß-D-GalNAc(4SO₄) could serve as an acceptor for GalNAc4S-6ST, thereby inhibiting GalNAc4S-6ST competitively. In this paper we compared the inhibitory effects of various glycosides in which various hydrophobic aglycons were attached to D-GalNAc(4SO₄) via ß anomeric configuration. p-Nitrophenyl-ß-D-GalNAc(4SO₄) and p-chlorophenyl-ß-D-GalNAc(4SO₄) were stronger inhibitors than phenyl-ß-D-GalNAc(4SO₄). Among inhibitors examined here, 3-estradiol-ß-D-GalNAc(4SO₄) was the strongest inhibitor; the Ki of 3-estradiol-ß-D-GalNAc(4SO₄) for the competitive inhibition was 0.008 mM, which was much lower than the Ki of phenyl-ß-D-GalNAc(4SO₄), 0.98 mM. In contrast, 7-estradiol-ß-D-GalNAc(4SO₄) showed only weak inhibition to GalNAc4S-6ST. 3-Estradiol- ß-D-GalNAc(4SO₄) did not inhibit chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase under the concentration where GalNAc4S-6ST was inhibited by 90%. When 3-estradiol- ß-D-GalNAc(4SO₄) was added to the culture medium of chondrosarcoma cells expressing human GalNAc4S-6ST, a significant, albeit small, reduction in the cellular synthesis of CS-E was observed. These results suggest that estradiol group of 3-estradiol-ß-D-GalNAc(4SO₄) may enhance the inhibitory activity of the glycoside through increasing the affinity to the enzyme and may allow the glycosides to diffuse at a low efficiency into the cells to inhibit cellular synthesis of CS-E.     

Insulin-like growth factor binding protein-1 levels are increased in patients with IgA nephropathy

The mechanisms underlying the pathogenesis of immunoglobulin A (IgA) nephropathy (IgAN) are not well understood. In this study, we examined gene expression profiles in kidneys obtained from mice with high serum IgA levels (HIGA mice), which exhibit features of human IgAN. Female inbred HIGA, established from the ddY line, were used in these experiments. Serum IgA levels, renal IgA deposition, mesangial proliferation, and glomerulosclerosis were increased in 32-week-old HIGA mice in comparison to ddY animals. By microarray analysis, five genes were observed to be increased by more than 2.5-fold in 32-week-old HIGA in comparison to 16-week-old HIGA; these same five genes were decreased more than 2.5-fold in 32-week-old ddY in comparison to 16-week-old ddY mice. Of these five genes, insulin-like growth factor (IGF) binding protein (IGFBP)-1 exhibited differential expression between these mouse lines, as confirmed by quantitative RT-PCR. In addition, serum IGFBP-1 levels were significantly higher in patients with IgAN than in healthy controls. In patients with IgAN, these levels correlated with measures of renal function, such as estimated glomerular filtration rate (eGFR), but not with sex, age, serum IgA, C3 levels, or IGF-1 levels. Pathologically, serum IGFBP-1 levels were significantly associated with the severity of renal injury, as assessed by mesangial cell proliferation and interstitial fibrosis. These results suggest that increased IGFBP-1 levels are associated with the severity of renal pathology in patients with IgAN.     

A novel role for a nodal-related protein; Xnr3 regulates convergent extension movements via the FGF receptor

Convergent extension behaviour is critical for the formation of the vertebrate body axis. In Xenopus, components of the Wnt signaling pathway have been shown to be required for convergent extension movements but the relationship between cell fate and morphogenesis is little understood. We show by loss of function analysis that Xnr3 activates Xbra expression through FGFR1. We show that eFGF activity is not essential in the pathway, and that dishevelled acts downstream of Xnr3 and not in a parallel pathway. We provide evidence for the involvement of the EGF-CFC protein FRL1, and suggest that the pro-domain of Xnr3 may be required for its activity. Since Xnr3 is a direct target of the maternal betacatenin/XTcf3 signaling pathway, it provides the link between the initial, maternally controlled, allocation of cell fate, and the morphogenetic movements of cells derived from the organizer.     

The Tobacco mosaic virus 126-kDa protein associated with virus replication and movement suppresses RNA silencing

Systemic symptoms induced on Nicotiana tabacum cv. Xanthi by Tobacco mosaic virus (TMV) are modulated by one or both amino-coterminal viral 126- and 183-kDa proteins: proteins involved in virus replication and cell-to-cell movement. Here we compare the systemic accumulation and gene silencing characteristics of TMV strains and mutants that express altered 126- and 183-kDa proteins and induce varying intensities of systemic symptoms on N. tabacum. Through grafting experiments, it was determined that M(IC)1,3, a mutant of the masked strain of TMV that accumulated locally and induced no systemic symptoms, moved through vascular tissue but failed to accumulate to high levels in systemic leaves. The lack of M(IC)1,3 accumulation in systemic leaves was correlated with RNA silencing activity in this tissue through the appearance of virus-specific, approximately 25-nucleotide RNAs and the loss of fluorescence from leaves of transgenic plants expressing the 126-kDa protein fused with green fluorescent protein (GFP). The ability of TMV strains and mutants altered in the 126-kDa protein open reading frame to cause systemic symptoms was positively correlated with their ability to transiently extend expression of the 126-kDa protein:GFP fusion and transiently suppress the silencing of free GFP in transgenic N. tabacum and transgenic N. benthamiana, respectively. Suppression of GFP silencing in N. benthamiana occurred only where virus accumulated to high levels. Using agroinfiltration assays, it was determined that the 126-kDa protein alone could delay GFP silencing. Based on these results and the known synergies between TMV and other viruses, the mechanism of suppression by the 126-kDa protein is compared with those utilized by other originally characterized suppressors of RNA silencing.     

Sodium spirulan as a potent inhibitor of arterial smooth muscle cell proliferation in vitro

Sodium spirulan (Na-SP) is a sulfated polysaccharide with M(r) approximately 220,000 isolated from the blue-green alga Spirulina platensis. The polysaccharide consists of two types of disaccharide repeating units, O-hexuronosyl-rhamnose (aldobiuronic acid) and O-rhamnosyl-3-O-methylrhamnose (acofriose) with sulfate groups, other minor saccharides and sodium ion. Since vascular smooth muscle cell proliferation is a crucial event in the progression of atherosclerosis, we investigated the effect of Na-SP on the proliferation of bovine arterial smooth muscle cells in culture. It was found that Na-SP markedly inhibits the proliferation without nonspecific cell damage. Either replacement of sodium ion with calcium ion or depolymerization of the Na-SP molecule to M(r) approximately 14,700 maintained the inhibitory activity, however, removal of sodium ion or desulfation markedly reduced the activity. Heparin and heparan sulfate also inhibited vascular smooth muscle cell growth but their effect was weaker than that of Na-SP; dextran sulfate, chondroitin sulfate, dermatan sulfate and hyaluronan failed to inhibit the cell growth. The present data suggest that Na-SP is a potent inhibitor of arterial smooth muscle cell proliferation, and the inhibitory effect requires a certain minimum sequence of polysaccharide structure whose molecular conformation is maintained by sodium ion bound to sulfate group.     

The Caenorhabditis elegans mRNA 5'-capping enzyme. In vitro and in vivo characterization

Eukaryotic mRNA capping enzymes are bifunctional, carrying both RNA triphosphatase (RTPase) and guanylyltransferase (GTase) activities. The Caenorhabditis elegans CEL-1 capping enzyme consists of an N-terminal region with RTPase activity and a C-terminal region that resembles known GTases, However, CEL-1 has not previously been shown to have GTase activity. Cloning of the cel-1 cDNA shows that the full-length protein has 623 amino acids, including an additional 38 residues at the C termini and 12 residues at the N termini not originally predicted from the genomic sequence. Full-length CEL-1 has RTPase and GTase activities, and the cDNA can functionally replace the capping enzyme genes in Saccharomyces cerevisiae. The CEL-1 RTPase domain is related by sequence to protein-tyrosine phosphatases; therefore, mutagenesis of residues predicted to be important for RTPase activity was carried out. CEL-1 uses a mechanism similar to protein-tyrosine phosphatases, except that there was not an absolute requirement for a conserved acidic residue that acts as a proton donor. CEL-1 shows a strong preference for RNA substrates of at least three nucleotides in length. RNA-mediated interference in C. elegans embryos shows that lack of CEL-1 causes development to arrest with a phenotype similar to that seen when RNA polymerase II elongation activity is disrupted. Therefore, capping is essential for gene expression in metazoans.     

Targeting and assembly of mitochondrial tail-anchored protein Tom5 to the TOM complex depend on a signal distinct from that of tail-anchored proteins dispersed in the membrane

Mitochondrial outer membrane proteins are synthesized without a cleavable presequence but instead contain segments responsible for mitochondrial targeting and membrane integration within the molecule: the transmembrane segment (TMS) and N- or C-terminal flanking segment. We analyzed targeting and integration of Tom5, a C-tail anchor protein associated with the preprotein translocase of the outer membrane, to the yeast mitochondrial outer membrane in vivo using green fluorescent protein as the reporter and compared the signal with other signals for proteins dispersed in the membrane. The functional assembly of Tom5 into the TOM complex was assessed by blue native PAGE and complementation of temperature-sensitive deltatom5 cells. Correct targeting and assembly required (i). an appropriate length TMS rather than hydrophobicity, (ii). a proline residue located at correct position in the TMS and specific residues near the proline, and (iii). that, in contrast to proteins dispersed in the outer membrane, the positive C-terminal segment was dispensable. Based on these findings, we constructed green fluorescent protein fusions with a C-terminal TMS in which the deduced sequences (minimum: Ser-Pro-Met) were inserted at an appropriate position within artificial Leu-Ala repeats. They were targeted to mitochondria and complemented the temperature-sensitive growth phenotype of deltatom5 yeast cells. The membrane-targeting mechanism of Tom5 appears to be distinct from that for proteins that are dispersed in the outer membrane.