Background: For the investigation of the molecular mechanisms involved in neurite outgrowth and differentiation, accurate and reproducible segmentation and quantification of neuronal processes are a prerequisite. To facilitate this task, we developed a semiautomatic neurite tracing technique. This article describes the design and validation of the technique. Methods:The technique was compared to fully manual delineation. Four observers repeatedly traced selected neurites in 20 fluorescence microscopy images of cells in culture, using both methods. Accuracy and reproducibility were determined by comparing the tracings to highresolution reference tracings, using two error measures. Labor intensiveness was measured in numbers of mouse clicks required. The significance of the results was determined by a Student t-test and by analysis of variance. Results: Both methods slightly underestimated the true neurite length, but the differences were not unanimously

Phosphorylation of ␣-synuclein (␣-syn) at Ser-129 is a hallmark of Parkinson disease and related synucleinopathies. However, the identity of the natural kinases and phosphatases responsible for regulating ␣-syn phosphorylation remain unknown. Here we demonstrate that three closely related members of the human Polo-like kinase (PLK) family (PLK1, PLK2, and PLK3) phosphorylate ␣-syn and ␤-syn specifically at Ser-129 and Ser-118, respectively. Unlike other kinases reported to partially phosphorylate ␣-syn at Ser-129 in vitro, phosphorylation by PLK2 and PLK3 is quantitative (>95% conversion). Only PLK1 and PLK3 phosphorylate ␤-syn at Ser-118, whereas no phosphorylation of ␥-syn was detected by any of the four PLKs (PLK1 to -4). PLK-mediated phosphorylation was greatly reduced in an isolated C-terminal fragment (residues 103-140) of ␣-syn, suggesting substrate recognition via the N-terminal repeats and/or the non-amyloid component domain of ␣-syn. PLKs specifically co-localized with phosphorylated Ser-129 (Ser(P)-129) ␣-syn in various subcellular compartments (cytoplasm, nucleus, and membranes) of mammalian cell lines and primary neurons as well as in ␣-syn transgenic mice, especially cortical brain areas involved in synaptic plasticity. Furthermore, we report that the levels of PLK2 are significantly increased in brains of Alzheimer disease and Lewy body disease patients. Taken together, these results provide biochemical and in vivo evidence of ␣-syn and ␤-syn phosphorylation by specific PLKs. Our results suggest a need for further studies to elucidate the potential role of PLK-syn interactions in the normal biology of these proteins as well as their involvement in the pathogenesis of Parkinson disease and other synucleinopathies.Increasing evidence suggests that phosphorylation may play an important role in the oligomerization and fibrillogenesis (1), Lewy body formation (1, 2) and neurotoxicity of ␣-synuclein (␣-syn) 5 in vivo (3). The majority of ␣-syn within Lewy bodies (LBs) in diseased human brains and animal models of Parkinson disease (PD) and related synucleinopathies is phosphorylated at Ser-129 (Ser(P)-129) (1, 2, 4 -7). Although recent studies support the notion that phosphorylation at Ser-129 is related to pathology and blocks ␣-syn fibrillization in vitro (8, 9), the exact mechanisms by which phosphorylation at Ser-129 modulates ␣-syn aggregation and toxicity in vivo remain elusive. Unraveling the role of phosphorylation in modulating the physiological and pathogenic activities of ␣-syn requires identification of the kinases and phosphatases involved in regulating its phosphorylation in vivo.Several kinases that phosphorylate ␣-syn at serine and tyrosine residues, primarily in its C-terminal region, have been identified using in vitro kinase assays and co-transfection studies. Casein kinase I and II, G-protein-coupled receptor kinases (GRK1, GRK2, GRK5, and GRK6), and calmodulin-dependent kinase II (10 -12) phosphorylate ␣-syn at Ser-129. Ser-87 is the only residue outside the C-terminal region report...

Biosynthesis of nitric oxide (NO) from L‐arginine modulates activity of iron‐dependent enzymes, including mitochondrial acontiase, an [Fe‐S] protein. We examined the effect of NO on the activity of iron regulatory factor (IRF), a cytoplasmic protein which modulates both ferritin mRNA translation and transferrin receptor mRNA stability by binding to specific mRNA sequences called iron responsive elements (IREs). Murine macrophages were activated with interferon‐gamma and lipopolysaccharide to induce NO synthase activity and cultured in the presence or absence of NG‐substituted analogues of L‐arginine which served as selective inhibitors of NO synthesis. Measurement of the nitrite concentration in the culture medium was taken as an index of NO production. Mitochondria‐free cytosols were then prepared and aconitase activity as well as IRE binding activity and induction of IRE binding activity were correlated and depended on NO synthesis after IFN‐gamma and/or LPS stimulation. Authentic NO gas as well as the NO‐generating compound 3‐morpholinosydnonimine (SIN‐1) also conversely modulated aconitase and IRE binding activities of purified recombinant IRF. These results provide evidence that endogenously produced NO may modulate the post‐transcriptional regulation of genes involved in iron homeostasis and support the hypothesis that the [Fe‐S] cluster of IRF mediates iron‐dependent regulation.

It has been proposed that p68, a nuclear protein of relative molecular mass 68,000, functions in the regulation of cell growth and division. A complementary DNA analysis of the protein has revealed extensive amino-acid sequence homology to the products of a set of genes recently identified in organisms as diverse as Escherichia coli and man, which include the eukaryotic translation initiation factor elF-4A. The protein products of the new gene family have several motifs in common which are thought to be involved in nucleic acid unwinding. As yet, however, only elF-4A, through its effect on RNA, has been shown to possess unwinding activity. Here we report that purified p68 also exhibits RNA-dependent ATPase activity and functions as an RNA helicase in vitro. The protein was first identified by its specific immunological cross reaction with the simian virus 40 large T antigen, the transforming protein of a small DNA tumour virus. Surprisingly, T antigen also has an RNA-unwinding activity: the homology between the two polypeptides, although confined to only a small region resembling the epitope of the cross-reacting antibody (PAb204), should therefore be of functional significance. Furthermore, the RNA-unwinding activity may be involved in the growth-regulating functions of both proteins.