Signaling through the B cell receptor (BCR) is essential for B cell function and development. Despite the key role of Syk in BCR signaling, little is known about the mechanism by which Syk transmits downstream effectors. BLNK (B cell LiNKer protein), a substrate for Syk, is now shown to be essential in activating phospholipase C (PLC)gamma 2 and JNK. The BCR-induced PLC gamma 2 activation, but not the JNK activation, was restored by introduction of PLC gamma 2 membrane-associated form into BLNK-deficient B cells. As JNK activation requires both Rac1 and PLC gamma 2, our results suggest that BLNK regulates the Rac1-JNK pathway, in addition to modulating PLC gamma 2 localization.

A fundamental question about the early pathogenesis of Alzheimer's disease (AD) concerns how toxic aggregates of amyloid ␤ protein (A␤) are formed from its nontoxic soluble form. We hypothesized previously that GM1 ganglioside-bound A␤ (GA␤) is involved in the process. We now examined this possibility using a novel monoclonal antibody raised against GA␤ purified from an AD brain. Here, we report that GA␤ has a conformation distinct from that of soluble A␤ and initiates A␤ aggregation by acting as a seed. Furthermore, GA␤ generation in the brain was validated by both immunohistochemical and immunoprecipitation studies. These results imply a mechanism underlying the onset of AD and suggest that an endogenous seed can be a target of therapeutic strategy.

Background: The Rho small GTPase regulates myosin II activity through the phosphorylation of the myosin light chain (MLC) by activating Rhokinase, which is a target of Rho. Several lines of evidence point to an important role of Rho in the action of lysophosphatidic acid (LPA) and thrombin in provoking neurite retraction in N1E-115 neuroblastoma cells.

PQBP-1 was isolated on the basis of its interaction with polyglutamine tracts. In this study, using in vitro and in vivo assays, we show that the association between ataxin-1 and PQBP-1 is positively influenced by expanded polyglutamine sequences. In cell lines, interaction between the two molecules induces apoptotic cell death. As a possible mechanism underlying this phenomenon, we found that mutant ataxin-1 enhances binding of PQBP-1 to the C-terminal domain of RNA polymerase II large subunit (Pol II). This reduces the level of phosphorylated Pol II and transcription. Our results suggest the involvement of PQBP-1 in the pathology of spinocerebellar ataxia type 1 (SCA1) and support the idea that modified transcription underlies polyglutamine-mediated pathology.

Upon DNA damage, p53-binding protein 1 (53BP1) relocalizes to sites of DNA double-strand breaks and forms discrete nuclear foci, suggesting its role in DNA damage responses. We show that 53BP1 changed its localization from the detergent soluble to insoluble fraction after treatment of cells with x-ray, but not with ultraviolet or hydroxyurea. Either DNase or phosphatase treatment of the insoluble fraction released 53BP1 into the soluble fraction, showing that 53BP1 binds to chromatin in a phosphorylation-dependent manner after X-irradiation of cells. 53BP1 was retained at discrete nuclear foci in X-irradiated cells even after detergent extraction of cells, showing that the chromatin binding of 53BP1 occurs at sites of DNA double-strand breaks. The minimal domain for focus formation was identified by immunofluorescence staining of cells ectopically expressed with 53BP1 deletion mutants. This domain consisted of conserved Tudor and Myb motifs. The Tudor plus Myb domain possessed chromatin binding activity in vivo and bound directly to both double-stranded and singlestranded DNA in vitro. This domain also stimulated endjoining by DNA ligase IV/Xrcc4, but not by T4 DNA ligase in vitro. We conclude that 53BP1 has the potential to participate directly in the repair of DNA double-strand breaks.

Bile acid-binding resins, such as cholestyramine and colestimide, have been clinically used as cholesterol-lowering agents. These agents bind bile acids in the intestine and reduce enterohepatic circulation of bile acids, leading to accelerated conversion of cholesterol to bile acids. A significant improvement in glycemic control was reported in patients with type 2 diabetes whose hyperlipidemia was treated with bile acid-binding resins. To confirm the effect of such drugs on glucose metabolism and to investigate the underlying mechanisms, an animal model of type 2 diabetes was given a high-fat diet with and without colestimide. Diet-induced obesity and fatty liver were markedly ameliorated by colestimide without decreasing the food intake. Hyperglycemia, insulin resistance, and insulin response to glucose, as well as dyslipidemia, were markedly and significantly ameliorated by the treatment. Gene expression of the liver indicated reduced expression of small heterodimer partner, a pleiotropic regulator of diverse metabolic pathways, as well as genes for both fatty acid synthesis and gluconeogenesis, by treatment with colestimide. This study provides a molecular basis for a link between bile acids and glucose metabolism and suggests the bile acid metabolism pathway as a novel therapeutic target for the treatment of obesity, insulin resistance, and type 2 diabetes. Diabetes 56:239 -247, 2007 T ype 2 diabetes and dyslipidemia are common metabolic disorders, and their worldwide prevalence is facing an acute increase, including a foreseen epidemic in diabetes with the number of diabetic individuals expected to more than double, reaching up to 300 million by 2025 (1). Type 2 diabetes and dyslipidemia are more frequently associated with each other than by chance, pointing to a possible common underlying mechanism(s) in their etiology (2). From the clinical point of view, dyslipidemia in patients with type 2 diabetes has several features: predominance of remnant particles and small dense LDL and elevation of plasma triglycerides, especially in a postprandial state, as well as low HDL cholesterol (3). These are highly atherogenic and, thus, predispose patients with diabetes to atherosclerotic disease, such as coronary artery disease and stroke, which not only accounts for 70% of mortality in patients with diabetes, but also places a social and economical burden in many countries (2-7). Therefore, therapeutic strategies that are beneficial for both conditions are strongly warranted.The liver plays a central role in systemic cholesterol metabolism and glucose homeostasis. Accumulating lines of evidence indicate the possible involvement of cholesterol metabolism in the liver, not only in the systemic lipid profile, but also in glucose homeostasis (8 -12), making hepatocellular cholesterol metabolism a key player in the pathogenesis of both dyslipidemia and hyperglycemia. Bile acids are major cholesterol metabolites that are synthesized in the liver and postprandially released into the small intestine. Most bile acids excret...

The small GTPase Rho is implicated in cytoskeletal rearrangements including stress fiber and focal adhesion formation and in the transcriptional activation of c-fos serum response element. In vitro, Rho-kinase, which is activated by Rho, phosphorylates not only myosin light chain (MLC) (thereby activating myosin ATPase) but also myosin phosphatase, thus inactivating myosin phosphatase. Rho-kinase is involved in the formation of stress fibers and focal adhesions in fibroblasts. Here we show that the expression of constitutively active Rho-kinase increased the level of MLC phosphorylation. The activity of Rho-kinase was necessary for maintaining the vinculin-containing focal adhesions, whereas organized actin stress fibers were not necessary for this. The microinjection of constitutively active Rho-kinase into fibroblasts induced the formation of focal adhesions to some extent under the conditions where organized actin stress fibers were disrupted. The expression of constitutively active Rhokinase also stimulated the transcriptional activity of c-fos serum response element. These results suggest that Rho-kinase has distinct roles in divergent pathways downstream of Rho, which include MLC phosphorylation leading to stress fiber formation, focal adhesion formation, and gene expression.

Activity-dependent changes in neuropsin gene expression in the hippocampus implies an involvement of neuropsin in neural plasticity. Since the deduced amino acid sequence of the gene contained the complete triplet (His-Asp-Ser) of the serine protease domain, the protein was postulated to have proteolytic activity. Recombinant full-length neuropsin produced in the baculovirus/ insect cell system was enzymatically inactive but was readily converted to active enzyme by endoprotease processing. The activational processing of prototype neuropsin involved the specific cleavage of the Lys 32 -Ile 33 bond near its N terminus. Native neuropsin that was purified with a purity of 1,100-fold from mouse brain had enzymatic characteristics identical to those of active-type recombinant neuropsin. Both brain and recombinant neuropsin had amidolytic activities cleaving Arg-X and Lys-X bonds in the synthetic chromogenic substrates, and the highest specific activity was found against Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide. The active-type recombinant neuropsin effectively cleaved fibronectin, an extracellular matrix protein. Taken together, these results indicate that this protease, which is enzymatically novel, has significant limbic effects by changing the extracellular matrix environment.Some proteases have been suggested to be related to neural cell dynamics in such processes as cell death, migration, cellto-cell adhesion and de-adhesion, process elongation, pathfinding, and axonal rearrangement (1-5). These phenomena have been investigated by supplying known proteases involved in blood coagulation, fibrinolysis, or digestion to neural cell cultures. However, the observations that the proteases are mainly localized in and released from non-neural cells do not support all of such neural effects (5-7). Thus, we postulated that neurons themselves may produce and release their own proteases to participate in the neural cell dynamics described above. Neuropsin (NP)1 was cloned from the mouse brain and was shown to be localized in mouse hippocampal pyramidal neurons (8). These results and the observation that its mRNA showed marked activity-dependent changes caused by plasticity-inducible stimuli are suggestive of some neural effects in limbic plasticity (8, 9). However, it is still not known whether NP protein has enzyme activity as suggested by the deduced amino acid sequence (8). We postulated that the enzyme activity might be a molecular basis for the physiological responses induced by various stimuli. Therefore, in the present study, we examined whether recombinant NP (r-NP) and brain NP had proteolytic activity against synthetic and natural substrates. EXPERIMENTAL PROCEDURESMaterials-Mono S, Sepharose 2B, CNBr-activated Sepharose 4B and CL-6B, Superdex-75HR, Superose 12, Resource S, and Protein G-Sepharose were from Amersham Pharmacia Biotech. Silver staining kits were from Bio-Rad. Diisopropyl fluorophosphate (DFP), benzamidine, bestatin, soybean trypsin inhibitor, human plasma thrombin (EC 3.4.4.13), and TNM-FH insect cel...