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Aquaporin-2 (AQP2) is essential to maintain body water homeostasis. AQP2 traffics from intracellular vesicles to the apical membrane of kidney collecting duct principal cells in response to vasopressin [arginine vasopressin (AVP)], a hormone released with low intravascular volume, which causes decreased kidney perfusion. Decreased kidney perfusion activates AMP-activated kinase (AMPK), a metabolic sensor that inhibits the activity of several transport proteins. We hypothesized that AMPK activation also inhibits AQP2 function. These putative AMPK effects could protect interstitial ionic gradients required for urinary concentration during metabolic stress when low intravascular volume induces AVP release. Here we found that short-term AMPK activation by treatment with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR; 75 min) in kidney tissue prevented baseline AQP2 apical accumulation in principal cells, but did not prevent AQP2 apical accumulation in response to the AVP analog desmopressin (dDAVP). Prolonged AMPK activation prevented AQP2 cell membrane accumulation in response to forskolin in mouse collecting duct mpkCCDc14cells. Moreover, AMPK inhibition accelerated hypotonic lysis of Xenopus oocytes expressing AQP2. We performed phosphorylation assays to elucidate the mechanism by which AMPK regulates AQP2. Although AMPK weakly phosphorylated immunoprecipitated AQP2 in vitro, no direct AMPK phosphorylation of the AQP2 COOH-terminus was detected by mass spectrometry. AMPK promoted Ser-261 phosphorylation and antagonized dDAVP-dependent phosphorylation of other AQP2 COOH-terminal sites in cells. Our findings suggest an increasing, time-dependent antagonism of AMPK on AQP2 regulation with AICAR-dependent inhibition of cAMP-dependent apical accumulation and AVP-dependent phosphorylation of AQP2. This inhibition likely occurs via a mechanism that does not involve direct AQP2 phosphorylation by AMPK.

Extracellular proton-secreting transport systems that contribute to extracellular pH include the vacuolar H+-ATPase (V-ATPase). This pump, which mediates ATP-driven transport of H+across membranes, is involved in metastasis. We previously showed (Alzamora R, Thali RF, Gong F, Smolak C, Li H, Baty CJ, Bertrand CA, Auchli Y, Brunisholz RA, Neumann D, Hallows KR, Pastor-Soler NM. J Biol Chem 285: 24676–24685, 2010) that V-ATPase A subunit phosphorylation at Ser-175 is important for PKA-induced V-ATPase activity at the membrane of kidney intercalated cells. However, Ser-175 is also located within a larger phosphorylation consensus sequence for Aurora kinases, which are known to phosphorylate proteins that contribute to the pathogenesis of metastatic carcinomas. We thus hypothesized that Aurora kinase A (AURKA), overexpressed in aggressive carcinomas, regulates the V-ATPase in human kidney carcinoma cells (Caki-2) via Ser-175 phosphorylation. We found that AURKA is abnormally expressed in Caki-2 cells, where it binds the V-ATPase A subunit in an AURKA phosphorylation-dependent manner. Treatment with the AURKA activator anacardic acid increased V-ATPase expression and activity at the plasma membrane of Caki-2 cells. In addition, AURKA phosphorylates the V-ATPase A subunit at Ser-175 in vitro and in Caki-2 cells. Immunolabeling revealed that anacardic acid induced marked membrane accumulation of the V-ATPase A subunit in transfected Caki-2 cells. However, anacardic acid failed to induce membrane accumulation of a phosphorylation-deficient Ser-175-to-Ala (S175A) A subunit mutant. Finally, S175A-expressing cells had decreased migration in a wound-healing assay compared with cells expressing wild-type or a phospho-mimetic Ser-175-to-Asp (S175D) mutant A subunit. We conclude that AURKA activates the V-ATPase in kidney carcinoma cells via phosphorylation of Ser-175 in the V-ATPase A subunit. This regulation contributes to kidney carcinoma V-ATPase-mediated extracellular acidification and cell migration.

Natural immunological tolerance can be induced in certain types of allogeneic liver transplantation in rats. To screen for genes associated with the induction of tolerance, suppression subtractive hybridization was performed in the rat liver transplantation model between a DA donor and PVG recipient combination where spontaneous immunological tolerance is known to occur without any immunosuppressive treatment. As a result, 112 genes were cloned from a DA liver graft that survived for 20 days in the fully allogeneic PVG recipient. After confirmation of the expression intensity using an in-house manufactured DNA array with cDNAs from the DA graft, 36 genes were classified in the highly expressed group and 26 moderately expressed group. In the first group, there were 8 immunoglobulin-related genes and 6 MHC class II-related genes, suggesting the existence of an underlying rejection response. Among those genes, an antiapoptotic gene in the p38 MAP kinase pathway, heme oxygenase gene (HO-1), and a ras cascade gene, IQ motif containing GTPase activating protein 1 (Iqgap1), retained biological significance. The results suggested that the molecular response to a liver graft tends to be antiapoptotic and to terminate the rejection response. Unfortunately, there was no gene identified that qualified as a putative immunosuppressive protein, liver suppressor factor-1 (LSF-1). The panel of genes identified in the present work will be a useful panel of candidate genes to investigate the induction of spontaneous tolerance.

International audience; Severe progressive neurological paediatric disease mucopolysaccharidosis III type C is caused by mutations in the HGSNAT gene leading to deficiency of acetyl-CoA: alpha-glucosaminide N-acetyltransferase involved in the lysosomal catabolism of heparan sulphate. To understand the pathophysiology of the disease we generated a mouse model of mucopolysaccharidosis III type C by germline inactivation of the Hgsnat gene. At 6-8 months mice showed hyperactivity, and reduced anxiety. Cognitive memory decline was detected at 10 months and at 12-13 months mice showed signs of unbalanced hesitant walk and urinary retention. Lysosomal accumulation of heparan sulphate was observed in hepatocytes, splenic sinus endothelium, cerebral microglia, liver Kupffer cells, fibroblasts and pericytes. Starting from 5 months, brain neurons showed enlarged, structurally abnormal mitochondria, impaired mitochondrial energy metabolism, and storage of densely packed autofluorescent material, gangliosides, lysozyme, phosphorylated tau, and amyloid-beta. Taken together, our data demonstrate for the first time that deficiency of acetyl-CoA: alpha-glucosaminide N-acetyltransferase causes lysosomal accumulation of heparan sulphate in microglial cells followed by their activation and cytokine release. They also show mitochondrial dysfunction in the neurons and neuronal loss explaining why mucopolysaccharidosis III type C manifests primarily as a neurodegenerative disease.

The cell cycle inhibitor p21 plays an important role in monocytic cell differentiation, during which it translocates from the nucleus to cytoplasm. This process involves the negative regulation of the p21 nuclear localization signal (NLS). Here, we sought to determine the relationship between the cytoplasmic translocation of p21 and another molecule, Brap2, a cytoplasmic protein which binds the NLS of BRCA1 and was recently reported to inactivate KSR in the Ras-activating signal pathway under the name of IMP. We report that p21 and Brap2 directly interact, both in vitro and in vivo, in a manner requiring the NLS of p21 and the C-terminal portion of Brap2. When it is cotransfected with Brap2, p21 is expressed in the cytoplasm. Monocytic differentiation of the promyelomonocytic cell lines U937 and HL60 is associated with the upregulation of Brap2 expression concomitantly with the upregulation and cytoplasmic relocalization of p21. Our results underscore the role played by Brap2 in the process of cytoplasmic translocation of p21 during monocyte differentiation.

Raft microdomains are glycolipid-enriched microdomain scaffolding molecules involved in signal transduction. The binding of Shiga toxin to globotriaosyl ceramide in raft microdomains of the human renal tubular cell line ACHN causes temporal activation of Src-kinase Yes. To study the downstream signaling mechanism proceeding to the activation of Yes, we raised monoclonal antibodies (MAbs) against raft microdomains. The MAbs were screened on the basis of, first, binding to raft microdomains with dot-blot immunostaining, second, intracellular localization of the epitope by flowcytometry after permeabilization, and third, translocation of the antigen molecules after Stx treatment by immunohistochemical staining. Raft.1 MAb bound to the molecules that accumulated to the particular region near the nucleus after Stx treatment. Two-dimensional Western blotting and matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis revealed that the antigen molecule is GTP binding protein beta subunits 1 and 2 (Gbeta1 and 2). That Raft.1 recognized Gbeta1 and 2 was further confirmed by the reactivity to recombinant Gbeta1 and 2 proteins. To our knowledge, this is the first report of production of a MAb recognizing Gbeta1 and 2. Because Gbeta1 and 2 are highly conserved all through organisms and are deeply involved in signal transduction, Raft.1 is expected to be utilized frequently in research.

Mucopolysaccharidosis type IIIB (MPS IIIB, Sanfilippo syndrome type B) is a lysosomal storage disease characterized by profound intellectual disability, dementia, and a lifespan of about two decades. The cause is mutation in the gene encoding α–N-acetylglucosaminidase (NAGLU), deficiency of NAGLU, and accumulation of heparan sulfate. Impediments to enzyme replacement therapy are the absence of mannose 6-phosphate on recombinant human NAGLU and the blood–brain barrier. To overcome the first impediment, a fusion protein of recombinant NAGLU and a fragment of insulin-like growth factor II (IGFII) was prepared for endocytosis by the mannose 6-phosphate/IGFII receptor. To bypass the blood–brain barrier, the fusion protein (“enzyme”) in artificial cerebrospinal fluid (“vehicle”) was administered intracerebroventricularly to the brain of adult MPS IIIB mice, four times over 2 wk. The brains were analyzed 1–28 d later and compared with brains of MPS IIIB mice that received vehicle alone or control (heterozygous) mice that received vehicle. There was marked uptake of the administered enzyme in many parts of the brain, where it persisted with a half-life of approximately 10 d. Heparan sulfate, and especially disease-specific heparan sulfate, was reduced to control level. A number of secondary accumulations in neurons [β-hexosaminidase, LAMP1(lysosome-associated membrane protein 1), SCMAS (subunit c of mitochondrial ATP synthase), glypican 5, β-amyloid, P-tau] were reduced almost to control level. CD68, a microglial protein, was reduced halfway. A large amount of enzyme also appeared in liver cells, where it reduced heparan sulfate and β-hexosaminidase accumulation to control levels. These results suggest the feasibility of enzyme replacement therapy for MPS IIIB.

To develop an in vitro experimental model of vascular smooth muscle cell hyperplasia, a major feature in chronic cardiac rejection, we studied a novel vascular smooth muscle cell line, P53LMAC01 (AC01), which was established from aortic smooth muscles of p53 knock-out mice, to determine its response to a platelet-derived growth factor (PDGF) and to Cyclosporin A (CsA). The responses were compared with those of human aortic smooth muscle cells (AOSMC). The AC01 exhibited a distinct proliferative response to PDGF similar to that of AOSMC under serum-free conditions. 10 ng/ml of PDGF-BB increased by a factor of 4.5 and PDGF-AB doubled the thymidine uptake, but PDGF-AA caused only a slight increase. The proliferation was markedly inhibited by 10(-6) M of CsA but less affected by 10(-7) M. These results indicate that the AC01 cell line could provide a convenient experimental system for investigating chronic rejection in vitro and that the system might work as a screening model of agents for treating transplant-related arteriosclerosis.

Cyclochlorotin (= chloropeptide, CC) is a hepatotoxic mycotoxin of Penicillium islandicum Sopp. The effect of CC on actin polymerization was examined by the measurement of changes in fluorescence intensity using pyrene-labeled actin and high shear viscosity. In the presence of CC, the time course of actin polymerization was accelerated in a dose dependent manner (2.5 ng/ml–2.5 μg/ml), without affecting the final level of viscosity. CC exerted a strong stabilizing effect on actin, enabling it to maintain its filamentous form in the presence of members of actin-binding proteins, including those of the gelsolin family prepared from hepatocytes. Microscopic observation revealed that in cultured hepatocytes, 1.0 μg/ml of CC induced bleb formation and changes in the microfilament. These observations indicated that after contact of the hepatocyte with CC, the following events were probable. The toxin passed through the cell membrane by a transport system and immediately reacted with the actin–actin binding proteins underlying the lipid bilayer. Bleb formation and hepatotoxicity were thus induced.

The binding of Shiga toxin (Stx) to Gb3Cer† in detergent-insoluble microdomains (DIM)/raft of the ACHN human renal tubular cell line causes the temporal activation of the Src-family kinase Yes [1]. As a strategy for examining signaling mechanisms in DIM/raft, monoclonal antibodies (MAbs) are reliable tools for characterizing the constituent molecules in these microdomains. Thus, we employed DIM/raft suspensions of ACHN cells as an immunogen to develop MAbs. Simply subcutaneous injections of ACHN DIM/raft could elevate the serum titer after several boosts. The first screening was performed using dot-blot immunostaining with culture supernatants on a polyvinylidene difluoride (PVDF) membrane, on which DIM/raft or their chloroform/methanol (C/M) (2:1, v/v) extracts were dot-blotted. The next screening was performed by flowcytometric analysis of ACHN cells treated with or without a permeabilizing reagent. Many of the clones (21/31 clones=68%) thus obtained were also found to recognize to lipid fractions of the DIM/raft. Strikingly, all of the 21 clones that reacted to the lipid fraction were found to recognize monosialosyl galactosylgloboside (MSGG) or GL7, which carries the SSEA-4 epitope. Using DIM/raft as immunogens may enable us to easily obtain MAbs for glycolipids.

The role of CD77 expressed on a fraction of germinal center B cells, also known as glycosphyngolipid Gb3, and as a functional receptor for Shiga toxins (Stx) in B-cell receptor (BCR)-mediated apoptosis was investigated. Using Stx1-sensitive Burkitt's lymphoma Ramos cells as an in vitro model of CD77(+) germinal center B cells, intracellular signaling events mediated by either Stx1 or anti-CD77 antibody were examined immunobiochemically and immunocytologically. We observed prompt activation of Lyn and Syk kinases leading to increased binding of these proteins to surface IgM (sIgM) in Ramos cells after Stx1 treatment. We also observed microscopic colocalization of CD77 and sIgM after stimulation with Stx1. Along with the synergism between the cross-linking of CD77 and that of sIgM in their effect on apoptosis induction, it was highly probable that CD77 cross-linking induces activation of the BCR signaling cascade. Analysis using sucrose density gradient centrifugation suggested that Stx1 binding to CD77 induced recruitment and activation of Lyn in the glycolipid-enriched membrane (GEM) fractions. Once activated, however, Lyn seemed to acquire an increased detergent solubility and moved outside of the GEM fractions. This study describes the participation of the GEM domain in BCR-signaling cascade and suggests a possible role of CD77 as a regulator of BCR-induced apoptosis in human B cells.

We pharmacologically studied the α1-adrenoceptor (AR) subtype(s) involved in receptor-mediated signalling in a novel vascular smooth muscle cell line cloned from p53 knockout mice, P53LMAC01 (AC01) cells. Radioligand binding studies with [125I]-HEAT showed the existence of a homogeneous population of binding site with an affinity (Kd value) of 0.4 nM and a maximum number of binding sites (Bmax) of 100 fmol mg−1 protein. Catecholamines competed for [125I]-HEAT binding stereospecifically and with the characteristic α1-AR potency series. Displacement curves for BMY-7378 and KMD-3213 best fitted a one-site model with a pKi value (−log10 (equilibrium inhibition constant)) of 6.06 and 7.07, respectively. Reverse transcription-polymerase chain reaction (RT–PCR) assay detected α1B- and α1D-AR, but not α1A-AR transcript. Chlorethylclonidine (CEC) treatment nearly abolished (−)noradrenaline (NA) (10 μM)-induced inositol[1,4,5]trisphosphate (IP3) production, and BMY-7378 inhibited the response with a Ki value of 0.3 nM, which value was similar to that obtained in the cells expressing α1D-AR. In both AC01 cells and cells expressing α1D-AR, BMY-7378 protected α1-ARs from CEC alkylation while it had little protective effect on CEC alkylation and NA-induced IP3 production in cells expressing α1B-AR. The results indicate that AC01 cells contain predominantly α1B-ARs and a small population of α1D-ARs; however, phosphoinositide (PI)/Ca2+ signalling is mainly mediated through the minor population of α1D-ARs, rather than the α1B-ARs. British Journal of Pharmacology (1999) 127, 756–762; doi:10.1038/sj.bjp.0702588

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder caused by the abnormal CAG triplet-repeat expansion resulting in an elongated polyglutamine (polyQ) stretch. We have recently showed that the DRPLA protein is cleaved during apoptosis by caspase-3, one of the cysteine protease family members known to be activated during apoptosis. We report here the subcellular localization of the DRPLA protein by fusing the green fluorescent protein as a tag. The full length DRPLA protein is localized predominantly but not exclusively in the nucleus regardless of the length of the polyQ stretch. In contrast, an N-terminal-deleted fragment containing polyQ produced by the proteolytic cleavage with caspase-3 is found both in the nucleus and the cytoplasm. Moreover, the same fragment with the elongated polyQ showed aggregation when overexpressed. Some cells with aggregate formation showed apoptotic phenotype. These findings raise the possibility that the DRPLA protein processed by caspase-3 may lead to aggregation of the protein resulting in the development of neurodegeneration.

Sanfilippo syndrome type B (mucopolysaccharidosis III B, MPS III B) is an autosomal recessive, neurodegenerative disease of children, characterized by profound mental retardation and dementia. The primary cause is mutation in the NAGLU gene, resulting in deficiency of α- N -acetylglucosaminidase and lysosomal accumulation of heparan sulfate. In the mouse model of MPS III B, neurons and microglia display the characteristic vacuolation of lysosomal storage of undegraded substrate, but neurons in the medial entorhinal cortex (MEC) display accumulation of several additional substances. We used whole genome microarray analysis to examine differential gene expression in MEC neurons isolated by laser capture microdissection from Naglu −/− and Naglu +/− mice. Neurons from the lateral entorhinal cortex (LEC) were used as tissue controls. The highest increase in gene expression (6- to 7-fold between mutant and control) in MEC and LEC neurons was that of Lyzs , which encodes lysozyme, but accumulation of lysozyme protein was seen in MEC neurons only. Because of a report that lysozyme induced the formation of hyperphosphorylated tau (P-tau) in cultured neurons, we searched for P-tau by immunohistochemistry. P-tau was found in MEC of Naglu −/− mice, in the same neurons as lysozyme. In older mutant mice, it was also seen in the dentate gyrus, an area important for memory. Electron microscopy of dentate gyrus neurons showed cytoplasmic inclusions of paired helical filaments, P-tau aggregates characteristic of tauopathies—a group of age-related dementias that include Alzheimer disease. Our findings indicate that the Sanfilippo syndrome type B should also be considered a tauopathy.

The interaction of reserpine with one of the cytoskeletal proteins, actin, was analyzed by the photoaffinity labelling method using [3H]reserpine. Reserpine bound sufficiently to G- or oligomeric actin, but hardly to F-actin under the same experimental conditions. This result could be explained if reserpine binds to a specific region of the G-actin molecule that is responsible for actin-actin interactions. It is concordant with this idea that [3H]reserpine bound only to specific proteolytic fragments of actin. When reserpine was mixed with crude extracts of two kinds of tissues, chicken gizzard smooth muscle and bovine adrenal medulla, it bound to the 42 kDa protein of sodium dodecyl sulfate-polyacrylamide gel electrophoresis in both cases. Its molecular size and abundance suggest strongly that this 42 kDa protein is actin. The binding of reserpine to actin was inhibited by dopamine in a dose-dependent manner. These results suggest that actin could be one of the target molecules of reserpine.

Summary In the growth arrested cultures of bovine carotid smooth muscle, K252a (10 – 100 ng/ml), a protein kinase inhibitor with wide spectrum suppressed the cell proliferation induced by TPA and increase of serum. K252a was more potent in the antiproliferative activity than H7, a C-kinase-specific inhibitor, but less than staurosporine, another wide-spectrum protein kinase inhibitor. Since C-kinase plays an important role in the signal transduction leading to the cell proliferation and K252a inhibits C-kinase in vitro , the antiproliferative effect of K252a to carotid smooth muscle cells is likely to be exerted through C-kinase dependent pathway.

The neurodegenerative disease MPS III B (Sanfilippo syndrome type B) is caused by mutations in the gene encoding the lysosomal enzyme α- N -acetylglucosaminidase, with a resulting block in heparan sulfate degradation. A mouse model with disruption of the Naglu gene allows detailed study of brain pathology. In contrast to somatic cells, which accumulate primarily heparan sulfate, neurons accumulate a number of apparently unrelated metabolites, including subunit c of mitochondrial ATP synthase (SCMAS). SCMAS accumulated from 1 month of age, primarily in the medial entorhinal cortex and layer V of the somatosensory cortex. Its accumulation was not due to the absence of specific proteases. Light microscopy of brain sections of 6-months-old mice showed SCMAS to accumulate in the same areas as glycosaminoglycan and unesterified cholesterol, in the same cells as ubiquitin and GM3 ganglioside, and in the same organelles as Lamp 1 and Lamp 2. Cryo-immuno electron microscopy showed SCMAS to be present in Lamp positive vesicles bounded by a single membrane (lysosomes), in fingerprint-like layered arrays. GM3 ganglioside was found in the same lysosomes, but was not associated with the SCMAS arrays. GM3 ganglioside was also seen in lysosomes of microglia, suggesting phagocytosis of neuronal membranes. Samples used for cryo-EM and further processed by standard EM procedures (osmium tetroxide fixation and plastic embedding) showed the disappearance of the SCMAS fingerprint arrays and appearance in the same location of "zebra bodies", well known but little understood inclusions in the brain of patients with mucopolysaccharidoses.

Shiga toxins (Stxs, also referred to as verotoxins) were first described as a novel cytotoxic activity against Vero cells. In this study, we report the characterization of an Stx1-resistant (R-) stock of Vero cells. (1) When the susceptibility of R-Vero cells to Stx1 cytotoxicity was compared to that of Stx1-sensitive (S-) Vero cells by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, cell viability after 48-hr exposure to 10 pg/ml of Stx1 was greater than 80% and less than 15%, respectively. (2) Although both a binding assay of fluorescence-labeled Stx1 and lipid analysis indicated considerable expression of Gb3Cer, a functional receptor for Stxs, in both Vero cells, anti-Gb3Cer monoclonal antibodies capable of binding to S-Vero cells failed to effectively label R-Vero cells, suggesting a conformational difference in the Gb3Cer expressed on R-Vero cells. (3) The lipid analysis also showed that the R-Vero cells contained significant amounts of Gb4Cer. In addition, introduction of exogenous Gb4Cer into S-Vero cells slightly inhibited Stx1 cytotoxicity, suggesting some correlation between glycosphingolipid composition and Stx1 resistance. (4) Both butyrate treatment and serum depression eliminated the Stx1 resistance of R-Vero cells. (5) The results of the analysis by confocal microscopy suggest a difference in intracellular transport of Stx1 between R-Vero and S-Vero cells. Further study of R-Vero cells may provide a model of Stx1 resistance via distinct intracellular transport of Stx1.

Mucopolysaccharidosis IIIB (MPS IIIB) is a lysosomal storage disorder caused by mutations in NAGLU , the gene encoding α- N -acetylglucosaminidase. The disease is characterized by profound mental retardation and eventual neurodegeneration, but relatively mild somatic manifestations. There is no available therapy. We have used a mouse knockout model of the disease to test therapy by genetically modified bone marrow. Bone marrow from Naglu −/− male mice was transduced with human NAGLU cDNA in an MND-MFG vector, and transplanted into 6- to 8-week-old lethally irradiated female −/− mice. Sham-treated mice received bone marrow transduced with eGFP cDNA in an MND vector. α- N -Acetylglucosaminidase activity in plasma and leukocytes, measured 3 and 6 months after transplantation, varied from marginal to nearly 30 times wild-type. A low level of α- N -acetylglucosaminidase activity, as little as provided by transplantation of unmodified Naglu +/+ bone marrow, could normalize biochemical defects (glycosaminoglycan storage and β-hexosaminidase elevation) in liver and spleen, but a very high level was required for an effect on kidney. Effects on the brain were best seen by examination of cellular morphology using light and electron microcopy. Mice that expressed very high levels of α- N -acetylglucosaminidase in blood had an increased number of normal-appearing neurons in the cortex and other parts of the brain, while microglia with engorged lysosomes had almost completely disappeared. Immunohistochemistry showed a marked decrease of staining for subunit c of mitochondrial ATP synthase and for Lamp1, markers of neuronal and microglial pathology, respectively, as well as a decrease in staining for glial fibrillary acid protein, a marker of activated astrocytes. These results show that genetically modified cells of hematopoietic origin can reduce the pathologic manifestations of MPS IIIB in the Naglu −/− mouse brain.

α- N -Acetylglucosaminidase deficiency (mucopolysaccharidosis IIIB, MPS IIIB) and α- l -iduronidase deficiency (MPS I) are heritable lysosomal storage diseases; neurodegeneration is prominent in MPS IIIB and in severe cases of MPS I. We have obtained morphologic and molecular evidence for the involvement of microglia in brain pathology of mouse models of the two diseases. In the cortex, a subset of microglia (sometimes perineuronal) consists of cells that are probably phagocytic; they have large storage vacuoles, react with MOMA-2 (monoclonal antibody against macrophages) and Griffonia simplicifolia isolectin IB 4 , and stain intensely for the lysosomal proteins Lamp-1, Lamp-2, and cathepsin D as well as for G M3 ganglioside. MOMA-2-positive cells appear at 1 and 6 months in MPS IIIB and MPS I mice, respectively, but though their number increases with age, they remain sparse. However, a profusion of cells carrying the macrophage CD68/macrosialin antigen appear in the cortex of both mouse models at 1 month. mRNA encoding CD68/macrosialin also increases at that time, as shown by microarray and Northern blot analyses. Ten other transcripts elevated in both mouse models are associated with macrophage functions, including complement C4, the three subunits of complement C1q, lysozyme M, cathepsins S and Z, cytochrome b558 small subunit, macrophage-specific protein 1, and DAP12. An increase in IFN-γ and IFN-γ receptor was observed by immunohistochemistry. These functional increases may represent activation of resident microglia, an influx and activation of blood monocytes, or both. They show an inflammatory component of brain disease in the two MPS, as is known for many neurodegenerative disorders.

Treatment for mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome type B) is hampered because recombinant alpha-N-acetylglucosaminidase (NAGLU) contains little/no mannose 6-phosphate. We developed a fusion protein of insulin-like growth factor 2 (IGF2) and NAGLU (rhNAGLU-IGF2). IGF2 is a natural ligand of the mannose-6phosphate receptor, and thus provides glycosylation-independent lysosomal targeting. Purified rhNAGLU-IGF2 is active in biochemical and cell-based assays (see Abstract: Aoyagi-Scharber, et al.). To evaluate rhNAGLU-IGF2 in vivo, adult (16 wk) MPS IIIB mice were catheterized into the left ventricle and given 4 twice-weekly injections of vehicle or rhNAGLU-IGF2 (100 μg). Mice were harvested 1d, 7d, 14d, and 28d after the last injection (n= 4-8 per group). Vehicle-treated heterozygotes were used as controls. Forebrain NAGLU activity ranged from 100x (1d, p b 0.001), 400x (7d, p = 0.001), 30x (14d, p = 0.9), and 130x fold (28d, p = 0.002) in rhNAGLU-IGF2 treated mice vs. vehicle-treated heterozygotes. Hexosaminidase activity was reduced by 26% (1d), 44% (7d), 51% (14d), and 57% (28d) in rhNAGLU-IGF2 treated vs vehicle-treated MPS IIIB mice (p b 0.001). Brain heparan sulfate was reduced from 228 pmol/ 5 μg brain (in vehicle-treated MPS IIIB) to 79.3 (1d), 24.8 (7d), 20.8 (14d), 17.4 (28d) in rhNAGLU-IGF2-treatedMPS IIIBmice (p b 0.001), vs 10.5 in vehicle-treated heterozygotes. In multiple brain regions, robust neuronal uptake was evident by immunostaining with NAGLU antibody and reduced LAMP1 signal with rhNAGLU-IGF2 treatment. In a separate experiment using 8 week-old mice, secondary accumulations were measured 1d after the last injection. Neuronal accumulations of SCMAS, glypican 5, amyloid beta and P-GSK3 beta in themedial entorhinal cortex and of P-tau in the dentate gyrus were reduced to the control level of these proteins. Accumulation of CD68 in activated microglia in the somatosensory cortex was reduced about half-way to control. In summary, we achieved broad distribution of NAGLU and improvement in neuropathology with short-term intraventricular rhNAGLU-IGF2. Glycosylation-independent lysosomal targeting may permit development of superior therapy for Sanfilippo syndrome type B. Support from NIH/NINDS 1R21NS078314-01A1 and BioMarin Pharmaceutical Inc.

Nitrobenzylthioinosine (NBTI), a nucleoside-transport inhibitor, has been found to possess the ability to prevent the cytotoxic action of Shiga toxin (Stx) 1 in human renal cortical epithelial cells (HRCECs), thereby protecting HRCECs from cell death. Further examination revealed that NBTI does not affect either the binding or the endocytosis of Stx1 but alters the intracellular transport of Stx1. Generally, endocytosed Stx1 is thought to be transported from endosomes to the endoplasmic reticulum. In NBTI-treated cells, however, the endocytosed Stx1 is delivered to an early endosome, but no further transportation occurs. Moreover, Stx1 is rapidly excreted from NBTI-treated HRCECs, preventing the accumulation of Stx1. Investigation of the NBTI-mediated protection mechanism against Stx cytotoxicity may provide insights into the analysis of Stx-mediated cell damage and lead to improvements in therapeutic approaches for diseases caused by Stx.

Objective In an attempt to clarify the megakaryo-specific regulatory mechanism of GPV gene transcription, we characterized the 5′-flanking region of the mouse GPV gene. Materials and Methods The promotor activity of a −481/+22 5′-fragment of the mouse GPV gene was examined in normal mouse bone marrow cells (BMC) and various human cell lines using two distinct reporter gene assay systems, luciferase and green fluorescence protein (GFP). Results When a DNA construct consisting of this fragment and a GFP reporter gene were transiently expressed in thrombopoietin-supported mouse BMC culture, GFP was identified only in megakaryocytes. The same construct expressed high levels of GFP in the human megakaryocytic Dami line. When assessed by dual luciferase assay, the full −481/+22 fragment could drive variable promoter activity in human as well as mouse megakaryocytic lines but did not work in non-megakaryocytic cells. Sufficient transcriptional activation of this fragment was restricted to the cells expressing apparent GPV mRNA. A deletion and point mutation study indicated that GATA and Ets motifs, typical cis-acting elements for platelet-specific genes, located of −75 and −46, respectively, were essential for promoter function. Conclusion The GPV promoter has the general characteristics found in platelet-specific genes, and the mechanism for megakaryocyte-specific, maturation-dependent regulation of GPV gene transcription is highly conserved between mouse and human. Analysis of GPV transcription mechanism utilizing human lines as well as BMC should provide new information on the final maturational process of megakaryocytes.

Smooth muscle myosin in the dephosphorylated state does not form filaments in vitro. However, thick filaments, which are composed of myosin and myosin-binding protein(s), persist in smooth muscle cells, even if myosin is subjected to the phosphorylation– dephosphorylation cycle. The characterization of telokin as a myosin-assembling protein successfully explained the discrepancy. However, smooth muscle cells that are devoid of telokin have been observed. We expected to find another ubiquitous protein with a similar role, and attempted to purify it from chicken gizzard. The 38k protein bound to both phosphorylated and dephosphorylated myosin to a similar extent. The effect of the myosin-binding activity was to assemble dephosphorylated myosin into filaments, although it had no effect on the phosphorylated myosin. The 38k protein bound to myosin with both COOH-terminal 20 and NH2-terminal 28 residues of the 38k protein being essential for myosin binding. The amino acid sequence of the 38k protein was not homologous to telokin, but to human p32, which was originally found in nuclei as a subunit of pre-mRNA splicing factor-2. Western blotting showed that the protein was expressed in various smooth muscles. Immunofluorescence microscopy with cultured smooth muscle cells revealed colocalization of the 38k protein with myosin and with other cytoskeletal elements. The absence of nuclear immunostaining was discussed in relation to smooth muscle differentiation.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neuro degrees enerative disorder associated with CAG/glutamine repeat expansion. While the DRPLA gene is ubiquitously expressed, neuron death occurs in specific anatomical areas of the brain. This predicts that the DRPLA protein interacts with other proteins and that these interactions may play a role in pathogenesis. Here, we describe a protein that binds to the DRPLA product. One of the clones isolated with a yeast two-hybrid system was identified as a human homolog of the insulin receptor tyrosine kinase substrate protein of 53 kDa (IRSp53). The gene produced two mRNA forms by differential splicing and encoded 552 and 521 amino acids, respectively. The longer form was mainly expressed in the brain and the shorter one in other tissues. The products were phosphorylated upon stimulation of cultured cells with insulin or insulin-like growth factor 1. Binding of the DRPLA protein to IRSp53 was ascertained by co-immunoprecipitation with antibodies and also by co-localization in perinuclear oval dots in cells expressing engineered constructs. A proline-rich region near the polyglutamine tract of the DRPLA protein and the SH3 domain of IRSp53 were involved in the binding. An extended polyglutamine tract significantly reduced binding ability in yeast cells, but not in in vitro binding assays. The identification of IRSp53 and other proteins detected by the yeast hybrid system predicts that DRPLA functions in a signal transduction pathway coupled with insulin/IGF-1.

We show here that the susceptibility of endothelial cells to Shiga toxin (Stx)s differs remarkably depending on their cellular origins. The concentration of Stx-1 required to reduce cell viability by 50% as measured by MTT assay was 30 and 300 fM for neonatal and adult human microvascular endothelial cells (HMVEC), respectively, and 30 pM for human coronary artery endothelial cells (HCAEC). Human umbilical venous endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC) showed no sensitivities to Stx-1. Surprisingly, Stx-2 was approximately 10–100 times more toxic to HMVEC than Stx-1. Moreover sodium butyrate sensitized HMVEC by 100-fold to the cytotoxic activity of Stxs. These results were found to reflect the amount of Gb3/CD77 on the cell surface on a per cell basis using flow cytometrical analysis. The high sensitivity of HMVEC to Stxs suggests their involvement in the pathogenesis of organ failure induced by Stx-producing Escherichia coli.

In skeletal muscle, excitation–contraction (E–C) coupling requires the conversion of the depolarization signal of the invaginated surface membrane, namely the transverse (T-) tubule, to Ca2+ release from the sarcoplasmic reticulum (SR). Signal transduction occurs at the junctional complex between the T-tubule and SR, designated as the triad junction, which contains two components essential for E–C coupling, namely the dihydropyridine receptor as the T-tubular voltage sensor and the ryanodine receptor as the SR Ca2+-release channel. However, functional expression of the two receptors seemed to constitute neither the signal-transduction system nor the junction between the surface and intracellular membranes in cultured cells, suggesting that some as-yet-unidentified molecules participate in both the machinery. In addition, the molecular basis of the formation of the triad junction is totally unknown. It is therefore important to examine the components localized to the triad junction. Here we report the identification using monoclonal antibody and primary structure by cDNA cloning of mitsugumin29, a novel transmembrane protein from the triad junction in skeletal muscle. This protein is homologous in amino acid sequence and shares characteristic structural features with the members of the synaptophysin family. The subcellular distribution and protein structure suggest that mitsugumin29 is involved in communication between the T-tubular and junctional SR membranes.

Here we report that we could obtain a highly differentiated smooth muscle cell line by screening the expression of a-smooth muscle actin from p53 knock out mice aorta. This cell revealed extended bipolar shape and expressed h-caldesmon and calponin as well as a-smooth muscle actin as protein markers of differentiated smooth muscle. Further intracellular calcium increase was induced by application of noradrenaline in a dose dependent manner and calcium oscillation was also observed in a higher dose (100μM). Appropriate application of 5-azacytidine enhanced these tendencies and induced slow contraction by endothelin-1 and phenylephrine.

The administration of 200 nM K252a to HuH7 suppressed the proliferation of the cells almost completely. The uptake of [3H]thymidine was inhibited, and flow cytometry revealed only one peak at 2C on day 3 after treatment with 100 nM K252a. The expression of proto-oncogene c-myc was not reduced. Despite the blockage at G1, both the size of the cells and the amount of cell protein had increased by 4 times by day 3 after treatment with K252a, while the cells secreted albumin and alpha-fetoprotein into the medium as usual. These results show that K252a can increase the cell size of HuH7 without losing its function by blocking the cell cycle at G1 phase.

K-252a, a protein kinase inhibitor with a wide spectrum of activity, inhibited the serum-stimulated proliferation of cultured bovine carotid endothelial cells dose-dependently, and all the cells became remarkably large, with a diameter of more than 150 μm at K-252a concentrations of 0.3-1 μg/ml. This effect of the agent was reproducible under the conditions described in this article. When the endothelial cells became abnormally large by K-252a, the surface area of the cell became wider, and the F-actin molecules increased in both number and length. Despite their abnormal size, K-252a-induced giant cells maintained at least three physiological functions characteristic to normal endothelial cells: 1) ability to take up acetylated low density lipoprotein, 2) ability to produce and secrete endothelin and 3) ability to respond via an increase of [Ca2+]i to the stimulation by bradykinin. These observations suggest that K-252a-induced giant cells are useful tools for examining the function of endothelial cells because it is very reproducible and can be produced by an easy treatment.

Brevin or plasma gelsolin, a calcium dependent actin-binding and actin-severing protein, was purified from bovine plasma by a very rapid and simple procedure; ammonium sulfate fractionation and only one step of anion exhange column chromatography by a convenient use. It takes only 24 hrs to complete all the procedure. The purity of brevin prepared by this method was more than 95 % on SDS-PAGE and total recovery was much better than previous preparation methods. This brevin preparation has about 8 isomers on 2-D PAGE and strong severing activity on F-actin under electron microscopic observation.

A Ca 2+ -dependent actin binding protein with a molecular weight of 74,000, was purified from bovine adrenal medulla by using deoxyribonuclease I affinity chromatography followed by ion-exchange chromatography and gel filtration. This protein broke actin filaments into fragments and promoted nucleation of actin polymerization in a Ca 2+ -dependent manner as effectively as gelsolin. Proteolytic and immunological comparison with gelsolin which is widely distributed actin-severing protein, indicated that the 74,000 mol. wt protein is a distinct protein, but its domain structure resembles that of gelsolin. Immunoblotting using antibody against this protein showed a tissue-specific distribution. The protein was detected in various endocrine, neuroendocrine and nervous tissues, but not in muscle tissues and plasma which contained relatively large amounts of cytoplasmic and plasma gelsolin. This fact might indicate that this actin-severing protein is involved in the regulation of the secretory process of endocrine and nervous tissues. In the exocytotic process regulated by Ca 2+ , this protein probably plays a role to free secretory organelles like vesicles from the cytoskeletal network, mainly F-actin, which prevents the movement of secretory vesicles in the resting state.

Top of pageAbstract Mucopolysaccharidosis IIIB (Sanfilippo syndrome type B) is a lysosomal storage disorder resulting from mutations in NAGLU, the gene encoding the enzyme alpha-N-acetylglucosaminidase that is required for the lysosomal degradation of heparan sulfate. The disease is characterized by profound mental retardation and eventual neurodegeneration, accompanied by relatively mild somatic manifestations. There is no available therapy. We have used a mouse knockout model of the disease to test gene therapy by genetically modified bone marrow. Bone marrow from Naglu −/− male mice was transduced with human NAGLU cDNA in an MND-MFG vector, and transplanted into 6–8 week-old lethally irradiated female −/− mice. Sham-treated mice received bone marrow transduced with eGFP cDNA in an MND vector. Analysis of alpha-N-acetylglucosaminidase in serum and white cells at 3 and 6 months after transplantation showed wide variability of expression, ranging from marginal to almost 30 times wild type. Biochemical studies showed that a fraction of normal alpha-N-acetylglucosaminidase activity (as little as provided by transplantation of unmodified +/+ bone marrow) could normalize biochemical defects (glycosaminoglycan storage and beta-hexosaminidase elevation) in liver and spleen. By contrast, only the highest level of activity had any effect on kidney and brain. Morphologic studies by light and electron microscopy showed a decreased number of vacuolated neurons and microglia in several areas of the brain, and an increased number of normal-appearing cells. Quantitation of the change in a section of the cortex is presented in the Table. Immunohistochemistry showed a marked reduction in staining for Lamp1 (a marker of storage in microglia) and for subunit c of ATP synthase (a marker of neuronal pathology). These results show that genetically modified cells of hematopoietic origin can reduce the pathologic manifestations in the brain of the mouse model of mucopolysaccharidosis IIIB.