Tau phosphorylation by cyclin-dependent kinase 5/p39 during brain development reduces its affinity for microtubules

The microtubule-associated protein tau is a developmentally regulated neuronal phosphoprotein. The phosphorylation of tau reduces its ability to bind and stabilize axonal microtubules during axonal growth. Although tau is phosphorylated by cyclin-dependent kinase 5 (Cdk5) in vitro, its in vivo roles remain unclear. Here, we show that tau is phosphorylated by Cdk5/p39 during brain development, resulting in a reduction of its affinity for microtubules. The activity of Cdk5 is tightly regulated by association with its neuronal activators, p35 or p39. The p35 and p39 expression levels were investigated in the developing mouse brain; the p39 expression level was higher in embryonic hind brain and spinal cord and in postnatal cerebral cortex, whereas that of p35 was most prominent in cerebral cortex at earlier stages of development. The ability of Cdk5 to phosphorylate tau was higher when in association with p39 than in association with p35. Tau phosphorylation at Ser-202 and Thr-205 was decreased in Cdk5-/- mouse brain but not in p35-/- mouse brain, suggesting that Cdk5/p39 is responsible for the in vivo phosphorylation of tau at these sites. Our data suggest that tau phosphorylation by Cdk5 may provide the neuronal microtubules with dynamic properties in a region-specific and developmentally regulated manner.     

Mechanism underlying the inner membrane retention of Escherichia coli lipoproteins caused by Lol avoidance signals

Escherichia coli lipoproteins are localized to either the inner or outer membrane depending on the residue at position 2. The inner membrane retention signal, Asp at position 2 in combination with certain residues at position 3, functions as a Lol avoidance signal, i.e. the signal inhibits the recognition of lipoproteins by LolCDE that releases lipoproteins from the inner membrane. To understand the role of the residue at position 2, outer membrane-specific lipoproteins with Cys at position 2 were subjected to chemical modification followed by the release reaction in reconstituted proteoliposomes. Sulfhydryl-specific introduction of nonprotein molecules or a negative charge to Cys did not inhibit the LolCDE-dependent release. In contrast, oxidation of Cys to cysteic acid resulted in generation of the Lol avoidance signal, indicating that the Lol avoidance signal requires a critical length of negative charge at the second residue. Furthermore, not only modification of the carboxylic acid of Asp at position 2 but also that of the amine of phosphatidylethanolamine abolished the Lol avoidance function. Based on these results, the Lol avoidance mechanism is discussed.     

Different protofilament-dependence of the microtubule binding between MAP2 and MAP4

To see a molecular basis of the difference in the microtubule binding between MAP2 and MAP4, we compared the binding of them onto microtubule and Zinc-sheet in the presence of various concentrations of NaCl. The Zinc-sheet is the lateral association of protofilaments arranged in an antiparallel fashion with alternatively exposed opposite surfaces, so that binding requiring adjacent protofilaments is restricted. While the salt-dependence of the MAP2 desorption was not altered between these tubulin polymers, MAP4 dissociated from Zinc-sheet at lower concentrations of NaCl than from microtubule. These results suggest that single protofilament is sufficient for microtubule binding of MAP2 as observed by Al-Bassam et al. [J. Cell Biol. 157 (2002) 1187], but MAP4 appeared to interact with adjacent protofilaments during microtubule-binding. Weakened binding on Zinc-sheets was also observed in the projection domain-deletion mutants of MAP4, so that the difference in the protofilament-dependence would lie in the relatively conserved microtubule-binding domain.     

TNF-alpha and IL-4 regulate expression of IL-13 receptor alpha2 on human fibroblasts

Two interleukin 13 receptors (IL-13Rs) have been identified as IL-13Ralpha1 and IL-13Ralpha2. IL-13Ralpha1 is composed of a heterodimer consisting of IL-13Ralpha1 and IL-4 receptor alpha (IL-4Ralpha) as a signaling subunit. In contrast, IL-13Ralpha2 is known as a decoy receptor for IL-13. In this study, we investigated the expression of IL-13Rs on human fibroblasts. IL-13Ralpha2 was significantly up-regulated after stimulation with tumor necrosis factor-alpha (TNF-alpha) and/or IL-4. In contrast, IL-13Ralpha1 was constitutively detectable and was not up-regulated. After the induction of IL-13alpha2 by IL-4, STAT6 phosphorylation through IL-13Ralpha1 by IL-13 was inhibited. We also detected large intracellular pools of IL-13Ralpha2 in fibroblasts quantitatively. Furthermore, mobilization of the IL-13Ralpha2 protein stores from the cytoplasm to the cell surface was prevented by an inhibitor of protein transport, brefeldin-A. These results indicate that TNF-alpha and IL-4 synergistically up-regulate the expression of IL-13Ralpha2 decoy receptor on human fibroblasts by inducing gene expression and mobilizing intracellular receptors, and thus may down-regulate the IL-13 signaling.     

Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors

The adult brain is extremely vulnerable to various insults. The recent discovery of neural progenitors in adult mammals, however, raises the possibility of repairing damaged tissue by recruiting their latent regenerative potential. Here we show that activation of endogenous progenitors leads to massive regeneration of hippocampal pyramidal neurons after ischemic brain injury. Endogenous progenitors proliferate in response to ischemia and subsequently migrate into the hippocampus to regenerate new neurons. Intraventricular infusion of growth factors markedly augments these responses, thereby increasing the number of newborn neurons. Our studies suggest that regenerated neurons are integrated into the existing brain circuitry and contribute to ameliorating neurological deficits. These results expand the possibility of novel neuronal cell regeneration therapies for stroke and other neurological diseases.     

Neuronal expression of macrophage colony stimulating factor in Purkinje cells and olfactory mitral cells of wild-type and cerebellar-mutant mice

Macrophage colony stimulating factor (M-CSF) is known to be the most effective growth factor for macrophage and microglial proliferation. In the brain tissue system, M-CSF is mainly produced in astrocytes and microglia, but is not known to occur in neurons. In the present paper, we examined the distribution of neurons expressing M-CSF in the mouse brain by immuno-histochemistry and in situ hybridization. We observed M-CSF immunoreactivity in both the cerebellum and the olfactory bulb. These positive cells were found to be Purkinje cells in the cerebellum, and mitral cells in the olfactory bulb. M-CSF mRNA expression was also confirmed to occur in these cells. Purkinje cells of reeler and weaver mutants showed M-CSF expression as seen in wild-type mice; however, those in the staggerer mutant did not. This expression in wild-type mice first appeared at postnatal day 7 and continued stably thereafter. When Purkinje cells were deprived of their climbing fibre innervation by inferior cerebellar pedunculotomy or by transplantation of cerebellar anlagen into the anterior eye chamber, the expression of M-CSF remained unchanged. These data indicate that expression of M-CSF in Purkinje cells is controlled by an intrinsic mechanism and could, therefore, be a new marker of postnatal development in rodent cerebella. The absence of M-CSF expression in the staggerer mutant is possibly due to developmental arrest in the early postnatal period.     

Identification of the sex pheromone components secreted by female moths of Peridroma saucia (Noctuidae: Noctuinae)

The variegated cutworm, Peridroma saucia Hübner, is a lepidopteran pest to a large number of crops in Canada, the United States, and Europe. It was probably naturalized in Japan in the 1970s. The pheromone glands of the female moth include two components with electroantennographic activity in a ratio of 3:1. GC-MS analyses of pheromone extracts untreated and treated with dimethyl disulfide revealed the major component to be (Z)-11-hexadecenyl acetate and the minor component to be (Z)-9-tetradecenyl acetate. The synthetic pheromone was used to attract a large number of males in a vegetable field in Tokyo, which suggests that this species has already become a harmful pest in Japan.     

Co-ordinating retinal histogenesis: early cell cycle exit enhances early cell fate determination in the Xenopus retina

The laminar arrays of distinct cell types in the vertebrate retina are built by a histogenic process in which cell fate is correlated with birth order. To explore this co-ordination mechanistically, we altered the relative timing of cell cycle exit in the developing Xenopus retina and asked whether this affected the activity of neural determinants. We found that Xath5, a bHLH proneural gene that promotes retinal ganglion cell (RGC) fate, ( Kanekar, S., Perron, M., Dorsky, R., Harris, W. A., Jan, L. Y., Jan, Y. N. and Vetter, M. L. (1997) Neuron 19, 981-994), does not cause these cells to be born prematurely. To drive cells out of the cell cycle early, therefore, we misexpressed the cyclin kinase inhibitor, p27Xic1. We found that early cell cycle exit potentiates the ability of Xath5 to promote RGC fate. Conversely, the cell cycle activator, cyclin E1, which inhibits cell cycle exit, biases Xath5-expressing cells toward later neuronal fates. We found that Notch activation in this system caused cells to exit the cell cycle prematurely, and when it is misexpressed with Xath5, it also potentiates the induction of RGCs. The potentiation is counteracted by co-expression of cyclin E1. These results suggest a model of histogenesis in which the activity of factors that promote early cell cycle exit enhances the activity of factors that promote early cellular fates.     

Detection of underlying characteristics of nuclear chromatin patterns of thyroid tumor cells using texture and factor analyses

BACKGROUND: Aspiration biopsy cytology of thyroid tumors has been used more frequently in recent times to differentiate between malignant and benign lesions. Chromatin patterns of the tumor cell nuclei are one of most important factors for cytologic diagnosis. The interpretation of nuclear chromatin patterns is subjective and more difficult than that of nuclear size or shape. In the present report, we investigated how to detect underlying chromatin characteristics of benign and malignant thyroid tumor cells by means of texture and factor analyses. METHODS: We employed a computer-aided system in which light microscopy was combined with an image processor and monochrome camera. Using this system, 100 randomly selected cells in a Papanicolaou stained, aspiration biopsy cytologic smear in each case of 39 benign and malignant thyroid tumor cases were digitized. We applied two-dimensional and higher texture analyses with the use of co-occurrence and run-length matrices to analyze the chromatin patterns. Factor analysis was used to determine whether a large number of independent variables actually measured one or more underlying common variables. RESULTS: According to parameters with high factor-loading values, the morphologic chromatin characters were classified into three categories according to heterogeneity, contrast, and homogeneity of chromatin patterns. On the basis of analyses with these morphologic categories, nuclei of papillary carcinoma showed higher contrast of chromatin patterns than did those of the benign group. Moreover, there was a variety of contrasting chromatin patterns among cells in each papillary carcinoma case in comparison with the benign group. In contrast, follicular carcinomas showed a significant difference in the standard deviation of factor 3, which indicated more monotonous chromatin patterns among cells in each follicular carcinoma case than in each benign case. CONCLUSION: We believe that this technique, using texture and factor analyses, is useful in the detection of underlying characteristics of nuclear chromatin patterns in aspiration biopsy cytology.