Altered patterns of DNA methylation associated with Helicobacter pylori (HP) infection of gastric epithelial cells are thought to contribute to gastric cancer risk. However, it is unclear whether this increased risk reflects an infection-associated inflammatory response or the infection itself. In this study, we sought to clarify mechanisms in a gerbil model of gastric cancer where we showed that HP infection is causally involved in induction of aberrant DNA methylation. By genome-wide screening, CpG islands that were aberrantly methylated in gerbil gastric cancer cell lines were isolated, and 10 islands were shown to be specifically methylated only in gastric mucosae infected with HP. By temporal analysis, methylation levels in gastric epithelial cells started to increase at 5 to 10 weeks after infection and reached high levels by 50 weeks. When HP was eradicated, methylation levels markedly decreased 10 and 20 weeks later, but they remained higher than those in gerbils that were not infected by HP. Expression levels of several inflammation-related genes (CXCL2, IL-1β, NOS2, and TNF-α) paralleled the temporal changes of methylation levels. Significantly suppressing inflammation with the immunosuppressive drug cyclosporin A did not affect colonization by HP but blocked the induction of altered DNA methylation. Our findings argue that DNA methylation alterations that occur in gastric mucosae after HP infection are composed of transient components and permanent components, and that it is the infection-associated inflammatory response, rather than HP itself, which is responsible for inducing the altered DNA methylation.

Rice (Oryza sativa) allelic sugary1 (sug1) mutants defective in isoamylase 1 (ISA1) accumulate varying levels of starch and phytoglycogen in their endosperm, and the activity of a pullulanase-type of a debranching enzyme (PUL) was found to correlate closely with the severity of the sug1 phenotype. Thus, three PUL-deficient mutants were generated to investigate the function of PUL in starch biosynthesis. The reduction of PUL activity had no pleiotropic effects on the other enzymes involved in starch biosynthesis. The short chains (DP ≤13) of amylopectin in PUL mutants were increased compared with that of the wild type, but the extent of the changes was much smaller than that of sug1 mutants. The α-glucan composition [amylose, amylopectin, water-soluble polysaccharide (WSP)] and the structure of the starch components (amylose and amylopectin) of the PUL mutants were essentially the same, although the average chain length of the B2-3 chains of amylopectin in the PUL mutant was ∼3 residues longer than that of the wild type. The double mutants between the PUL-null and mild sug1 mutants still retained starch in the outer layer of endosperm tissue, while the amounts of WSP and short chains (DP ≤7) of amylopectin were higher than those of the sug1 mutant; this indicates that the PUL function partially overlaps with that of ISA1 and its deficiency has a much smaller effect on the synthesis of amylopectin than ISA1 deficiency and the variation of the sug1 phenotype is not significantly dependent on the PUL activities.

Mitogen-activated protein kinase (MAPK), a serinelthreonine-specific protein kinase which is generally activated by stimulation with various growth factors and phorbol esters, utilizes microtubule-associated protein (MAP) 2 as a good substrate in vitro. We have found that MAPKcatalyzed phosphorylation of MAP2 resulted in a significant loss in its ability to induce tubulin polymerization. The chymotryptic fragments, containing a microtubule-binding domain of MAP2, were phosphorylated by MAPK and the ability of the fragments to induce tubulin polymerization was also greatly decreased by the phosphorylation, suggesting that phosphorylation of the microtubulebinding domain is important for functional alteration of MAP2. In addition to MAP2, a 190-kDa heat-stable MAP (MAP4) found in various tissues and cells, was a good substrate for MAPK in vitro. Phosphorylation of MAP4 inactivated tubulin polymerization. We examined the effect of phosphorylation of MAP2 and MAP4 on the dynamics of microtubules nucleated by purified centrosomes in vitro. The data showed that MAPK-catalyzed phosphorylation of MAP2 and MAP4 reduced their ability to increase the apparent elongation rate and the number of microtubules nucleated by the centrosome. Thus, MAPK is capable of phosphorylating MAPS and negatively regulating their microtubule-stabilizing function.Mitogen-activated protein kinase (MAPK) [l -101, a serinelthreonine-specific protein kinase which utilizes microtubule-associated protein (MAP) 2 and myelin basic protein as major substrates in v i m , is universally activated in quiescent fibroblast cells by a variety of mitogens, including epidermal growth factor (EGF), platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor-I, insulin and 12-0-tetradecanoyl-phorbol13-acetate. Although in vivo substrates for MAPK have been largely unknown, S6 kinase is considered to be a potential target for MAPK, since MAPK has been reported to phosphorylate and activate S6 kinase in vitvo [4, 6,Sl. Our studies on MAPK were originally derived from the observation that various mitogens stimulated, in general, phosphorylation of MAPl-like and MAP2-like proteins in quiescent cells [ll, 121. It is possible that these MAP-like proteins could be phosphorylated by the MAPKmediated pathway in cells.MAP2 is phosphorylated in vitro by CAMP-dependent protein kinase [13 -171, Ca2 '/calmodulin-dependent protein kinase I1 [18-201, protein kinases results equally in reduction of its tubulin-polymerizing ability, probably due to phosphorylation of the microtubulebinding domain of MAP2, although the projection domain could also be phosphorylated by these kinases [14, 17, 20, 25, 261. Silliman and Sturgill (1989) EXPERIMENTAL PROCEDURES Preparation of cytoskeletal proteinsMAP2 and tubulin were purified from porcine brains as previously described [24 -261. An adrenal 1 90-kDa MAP (MAP4) was purified from bovine adrenal cortex as described previously 1331. Purification of the microtubule-binding fragments of MAP2The chymotryptic frag...

Aberrant DNA methylation is associated with many types of human cancers. To identify genes silenced in human colorectal cancers, we performed a microarray analysis for genes whose expression was induced by treatment of HCT116 human colon cancer cells with a demethylating agent, 5-aza-2 0 -deoxycitidine (5-aza-dC). Seven known genes were identified as being upregulated (8-fold) and expressed at more than twice as high as the average level. Among these was the UCHL1 gene (also known as PGP9.5), which is involved in regulation of cellular ubiquitin levels. A dense CpG island in its promoter region was completely methylated in HCT116 cells, and no mRNA was detected. 5-Aza-dC treatment of HCT116 cells induced dose-dependent demethylation of the CpG island, and restored UCHL1 mRNA and protein expression. UCHL1 silencing was observed in 11 of 12 human colorectal cancer cell lines, and its methylation was detected in 8 of 17 primary colorectal cancers. Further, UCHL1 silencing was observed in 6 of 13 ovarian cancer cell lines, and its methylation was detected in 1 of 17 primary ovarian cancers. These results showed that UCHL1 is inactivated in human colorectal and ovarian cancers by its promoter methylation, and suggest that disturbance of cellular ubiquitin levels is present.

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Glycogen synthase kinase 3 (GSK3) has been identified as a promising target for the treatment of Alzheimer’s disease (AD), where abnormal activation of this enzyme has been associated with hyperphosphorylation of tau proteins. This study describes the effects of the selective GSK3 inhibitor, SAR502250, in models of neuroprotection and neuropsychiatric symptoms (NPS) associated with AD. In P301L human tau transgenic mice, SAR502250 attenuated tau hyperphosphorylation in the cortex and spinal cord. SAR502250 prevented the increase in neuronal cell death in rat embryonic hippocampal neurons following application of the neurotoxic peptide, Aβ25–35. In behavioral studies, SAR502250 improved the cognitive deficit in aged transgenic APP(SW)/Tau(VLW) mice or in adult mice after infusion of Aβ25–35. It attenuated aggression in the mouse defense test battery and improved depressive-like state of mice in the chronic mild stress procedure after 4 weeks of treatment. Moreover, SAR502250 decreased hyperactivity produced by psychostimulants. In contrast, the drug failed to modify anxiety-related behaviors or sensorimotor gating deficit. This profile confirms the neuroprotective effects of GSK3 inhibitors and suggests an additional potential in the treatment of some NPS associated with AD.

Topical therapy is the most favored form of treatment for whitening against hyperpigmentation and sunburn because it lends itself to self-administration, patient compliance, and absence of systemic adverse effects. However, transdermal delivery of hydrophilic chemicals is difficult. The main purpose of this study is to develop a delivering system of hydrophilic drugs and proteins across the skin. Hydroquinone (HQ), a well-known tyrosinase inhibitor and antimelanogenesis compound, and enhanced green fluorescent protein (EGFP) were fused with eleven poly-arginine (11R). Both HQ-11R and EGFP-11R were efficiently delivered in B16 cells, a mouse melanoma cell line. HQ-11R was as effective as HQ alone at inhibiting melanin synthesis in B16 cells. EGFP-11R was efficiently delivered into cells of the epidermis with 4-(1-pyrenyl)-butyric acid (PB), a counteranion bearing an aromatic hydrophobic moiety, in vivo, but EGFP alone or EGFP-11R without PB was not. Finally, topical application of HQ-11R with PB significantly inhibited UV irradiationinduced pigmentation in guinea pigs compared with HQ alone. These results suggest that topical therapy using poly-arginine in combination with PB is useful for the delivery of hydrophilic drugs and proteins by the transdermal route . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Abstract Topical therapy is the most favored form of treatment for whitening against hyperpigmentation and sunburn because it lends itself to self-administration, patient compliance, and absence of systemic adverse effects. However, transdermal delivery of hydrophilic chemicals is difficult. The main purpose of this study is to develop a delivering system of hydrophilic drugs and proteins across the skin. Hydroquinone (HQ), a well-known tyrosinase inhibitor and antimelanogenesis compound, and enhanced green fluorescent protein (EGFP) were fused with eleven poly-arginine (11R).Both HQ-11R and EGFP-11R were efficiently delivered in B16 cells, a mouse melanoma cell line. HQ-11R was as effective as HQ alone at inhibiting melanin synthesis in B16 cells. EGFP-11R was efficiently delivered into cells of the epidermis with 4-(1-pyrenyl)-butyric acid (PB), a counteranion bearing an aromatic hydrophobic moiety, in vivo, but EGFP alone or EGFP-11R without PB was not. Finally, topical application of HQ-11R with PB significantly inhibited UV irradiation-induced pigmentation in guinea pigs compared with HQ alone. These results suggest that topical therapy using poly-arginine in combination with PB is useful for the delivery of hydrophilic drugs and proteins by the transdermal route. *Abstract1 2 3 4 5 6 7 8

We have examined the phosphorylation of bovine microtubule-associated protein 4 (MAP4), formerly named MAP-U, by protein kinase C (PKC). When MAP4 was incubated with PKC, about 1 mol of phosphate was incorporated/mol of MAP4. Phosphorylation of MAP4 caused a remarkable decrease in the ability of the MAP to stimulate microtubule assembly. MAP4 consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain. The carboxyl-terminal domain is subdivided into a Pro-rich region and an assembly-promoting (AP) sequence region containing four tandem repeats of AP sequence that is conserved in MAP4, MAP2, and tau [Aizawa et al. (1990) J. Biol. Chem. 265, 13849-13855]. In order to identify the site of MAP4 phosphorylated by PKC, a series of expressed MAP4 fragments was prepared and treated with the kinase. A fragment corresponding to the Pro-rich region (P fragment) was phosphorylated, while fragments corresponding to the projection domain and the AP sequence region were not. In addition, chymotryptic digestion of an authentic MAP4 prephosphorylated by PKC revealed that phosphate was incorporated almost exclusively into a 27-kDa fragment containing the carboxyl-terminal half of the Pro-rich region. We investigated the phosphorylation site in MAP4 using the P fragment and found that Ser815 was phosphorylated almost exclusively. We conclude that the phosphorylation of a single Ser residue in the Pro-rich region negatively regulates the assembly-promoting activity of MAP4.