Coherence between oscillations in the cardiorespiratory system and tissue oxygen index in muscle recovering from intensive exercise in humans.

It has been shown that the tissue oxygen index (TOI) measured by near-infrared spectroscopy oscillates at very low frequencies during recovery after exercise and that this oscillation is derived from interactions among biochemical substances involved in oxidative metabolism in skeletal muscle. As a further step, we examined whether TOI in muscle interacts through oscillation with factors related to oxygen in the cardiorespiratory system. For this examination, coherence and phase difference between the TOI in the vastus lateralis and heart rate (HR) and between TOI and arterial oxygen saturation (SpO2) were sequentially determined during recovery (2-60 min) after severe cycle exercise with a workload of 7.5% of body weight for 20 s. Significant coherence between TOI and HR was obtained in the very low-frequency band (approximate range: 0.002-0.03 Hz) and in the low-frequency band (approximate range: 0.06-0.12 Hz). The phase difference was negative in the low-frequency band and positive in the very low-frequency band. The coherence between TOI and SpO2 was significant in the very low-frequency band. The phase difference was negative. There were no sequential changes in these coherences and phase differences. The results suggest that TOI in skeletal muscle interrelates with factors related to the heart and lungs.

Phosphorylation of neuronal nitric oxide synthase at Ser847 in the nucleus intermediolateralis after spinal cord injury in mice.

We previously demonstrated that Ca2+/calmodulin (CaM)-dependent protein kinase IIalpha (CaM-KIIalpha) can phosphorylate neuronal nitric oxide synthase (nNOS) at Ser847 and attenuate NOS activity in neuronal cells. In the present study we focused on chronological alteration in levels and cellular location of nNOS, phosphorylated (p)-Ser847-nNOS (NP847), CaM-KII and p-Thr286-CaM-KIIalpha following spinal cord injury (SCI) in mice. Western blot analysis showed nNOS to be significantly phosphorylated at Ser847 from 3 h after SCI, peaking at 24 h and gradually decreasing thereafter, and CaM-KII to be colocalized with nNOS after SCI. Immunohistochemical analysis revealed that SCI causes an increase in both NP847 and p-Thr286-CaM-KIIalpha in the nucleus intermediolateralis. These findings suggest that SCI induces p-Thr286-CaM-KIIalpha, which phosphorylates the nNOS at Ser847 in the nucleus intermediolateralis where NO is thought to play a role as a neurotransmitter in autonomic preganglionic neurons. Thus, the NP847 signaling pathway might be involved in the autonomic failure which occurs immediately after SCI.

A Novel Phase-contrast Transmission Electron Microscopy Producing High-contrast Topographic Images of Weak objects.

We report a novel class of transmission electron microscope (TEM), the difference-contrast electron microscope (DTEM), which displays nanostructures of thin specimen objects in a topographical manner. Topography obtained by the difference-contrast develops shadowgraphs in pseudo three-dimension, namely volume-like representation of projected objects as if things are illuminated by light from one direction. The specific optical device tomanipulate electron waves for DTEM is the hemicircular π phase-plate, which appears to be quite distinguishable from the Zernike phase plate utilized in Zernike phase-contrast TEM, while both have to be placed onto the back-focal plane of the objective lens. The topographic images obtained with DTEM for ultrathin sections of kidney cells were compared with those obtained with conventional TEM. DTEM confirmed the experimental advantage of high contrast topography by visualizing ultrastructural details inside the cells.

Temperature-sensitive phenotype of Chinese hamster ovary cells defective in PEX5 gene.

SK32 mutant cells, which were isolated as peroxisome-deficient Chinese hamster ovary (CHO) cells by an advantage of a visible peroxisome form of green fluorescent protein (GFP), were found to suffer from a functional loss of PEX5 gene encoding for PTS1R. The sequence analysis of cDNA indicated that PEX5 gene encoded for the two isoforms composed of 603 amino acids (PTS1RS) and 640 amino acids (PTS1RL). The mutation changed glycine to arginine at amino acid position 343 of PTS1RL (corresponding to the position 306 of PTS1RS) in SK32 cells. The mutant cells exhibited a temperature-sensitive (TS) phenotype on the peroxisomal localizations of the recombinant GFP and urate oxidase appending a genuine peroxisome targeting signal 1 (PTS1), a tripeptide of Ser-Lys-Leu (SKL) at the C-terminus, but did not on that of catalase harboring a divergent PTS1, Lys-Ala-Asn-Leu (KANL) sequence. 3-ketoacyl-CoA thiolase (hereafter referred to as thiolase), which harbors an extension sequence (PTS2) at the N-terminus, never appeared to be affected on the peroxisomal localization in the mutant cells. When thiolase was examined on the molecular size in the mutant cells, the enzyme existed as the larger precursor form in the peroxisomes at 37 degrees C and a considerable part (almost half) was converted to the mature size at 30 degrees C. These results indicate that the amino acid substitution, Gly306Arg in PTS1RS and/or Gly343Arg in PTSRL, gives rise to TS phenotype on the peroxisomal translocation of PTS1 proteins and the maturation of PTS2 protein.

Role of peroxisome proliferator-activated receptor gamma and its ligands in non-neoplastic and neoplastic human urothelial cells.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily of ligand-activated transcription factors and is expressed in several types of tissue. Although PPARgamma reportedly is expressed in normal urothelium, its function is unknown. We examined the expression of PPARgamma in normal urothelium and bladder cancer in an attempt to assess its functional role. Immunohistochemical staining revealed normal urothelium to express PPARgamma uniformly. All low-grade carcinomas were positive either diffusely or focally, whereas staining was primarily focal or absent in high-grade carcinomas. A nonneoplastic urothelial cell line (1T-1), a low-grade (RT4) carcinoma cell line, and two high-grade (T24 and 253J) carcinoma cell lines in culture expressed PPARgamma mRNA and protein. Luciferase assay indicated that PPARgamma was functional. PPARgamma ligands (15-deoxy-Delta(12,14)-prostaglandin J(2), troglitazone and pioglitazone) suppressed the growth of nonneoplastic and neoplastic urothelial cells in a dose-dependent manner. However, neoplastic cells were more resistant than were nonneoplastic cells. Failure to express PPARgamma or ineffective transcriptional activity may be some of the mechanisms responsible for resistance to the inhibitory action of PPARgamma ligands.

High serum IgG4 concentrations in patients with sclerosing pancreatitis.

BACKGROUND: Sclerosing pancreatitis is a unique form of pancreatitis that is characterized by irregular narrowing of the main pancreatic duct, lymphoplasmacytic inflammation of the pancreas, and hypergammaglobulinemia and that responds to glucocorticoid treatment. Preliminary studies suggested that serum IgG4 concentrations are elevated in this disease but not in other diseases of the pancreas or biliary tract. METHODS: We measured serum IgG4 concentrations using single radial immunodiffusion and an enzyme-linked immunosorbent assay in 20 patients with sclerosing pancreatitis, 20 age- and sex-matched normal subjects, and 154 patients with pancreatic cancer, ordinary chronic pancreatitis, primary biliary cirrhosis, primary sclerosing cholangitis, or Sjögren's syndrome. Serum concentrations of immune complexes and the IgG4 subclass of immune complexes were determined by means of an enzyme-linked immunosorbent assay with monoclonal rheumatoid factor. RESULTS: The median serum IgG4 concentration in the patients with sclerosing pancreatitis was 663 mg per deciliter (5th and 95th percentiles, 136 and 1150), as compared with 51 mg per deciliter (5th and 95th percentiles, 15 and 128) in normal subjects (P<0.001). The serum IgG4 concentrations in the other groups of patients were similar to those in the normal subjects. In patients with sclerosing pancreatitis, serum concentrations of immune complexes and the IgG4 subclass of immune complexes were significantly higher before glucocorticoid therapy than after four weeks of such therapy. Glucocorticoid therapy induced clinical remissions and significantly decreased serum concentrations of IgG4, immune complexes, and the IgG4 subclass of immune complexes. CONCLUSIONS: Patients with sclerosing pancreatitis have high serum IgG4 concentrations, providing a useful means of distinguishing this disorder from other diseases of the pancreas or biliary tract.

Localization of calcineurin in the mature and developing retina.

We studied the localization of calcineurin by immunoblotting analysis and immunohistochemistry as a first step in clarifying the role of calcineurin in the retina. Rat, bovine, and human retinal tissues were examined with subtype-nonspecific and subtype-specific antibodies for the A alpha and A beta isoforms of its catalytic subunit. In mature retinas of the three species, calcineurin was localized mainly in the cell bodies of ganglion cells and the cells in the inner nuclear layer, in which amacrine cells were distinctively positive. The calcineurin A alpha and A beta isoforms were differentially localized in the nucleus and the cytoplasm of the ganglion cell, respectively. Calcineurin was also present in developing rat retinas, in which the ganglion cells were consistently positive for it. The presence of calcineurin across mammalian species and regardless of age shown in the present study may reflect its importance in visual function and retinal development, although its function in the retina has not yet been clarified. (J Histochem Cytochem 49:187-195, 2001)

Occurrence of a transcription factor, signal transducer and activators of transcription 3 (Stat3), in the postsynaptic density of the rat brain.

Distribution of a signal transducer and activators of transcription 3 (Stat3) was examined in the rat brain. Immunoreactivity was distributed in the neurons, as well as glia cells, throughout the rat brain. Western blotting of subcellular fractions showed distribution in the synaptic fractions (synaptosome, synaptic plasma membrane, and postsynaptic density, PSD). The occurrence of Stat3 in the synaptic site, especially in the PSD, was confirmed by immunoelectron microscopic examination. The PSD fraction had an activity that phosphorylated Stat3 at the tyrosine-705 site, which was confirmed by both Western blotting and immunoprecipitation. The PSD fraction also had a janus kinase 2 (Jak2)-like molecule, Jak2, believed to phosphorylate Stat3. These results indicate that the Jak2/Stat3 signaling system, a major pathway of cytokine signaling in the immune response system, may also play a role at the postsynaptic sites.

Sex-dependent regulation of hepatic peroxisome proliferation in mice by trichloroethylene via peroxisome proliferator-activated receptor alpha (PPARalpha).

The mechanism of trichloroethylene-induced liver peroxisome proliferation and the sex difference in response was investigated using wild-type Sv/129 and peroxisome proliferator-activated receptor alpha (PPARalpha)-null mice. Trichloroethylene treatment (0.75 g/kg for 2 weeks by gavage) resulted in liver peroxisome proliferation in wild-type mice, but not in PPARalpha-null mice, suggesting that trichloroethylene-induced peroxisome proliferation is primarily mediated by PPARalpha. No remarkable sex difference was observed in induction of peroxisome proliferation, as measured morphologically, but a markedly higher induction of several enzymes and PPARalpha protein and mRNA was found in males. On the other hand, trichloroethylene induced liver cytochrome P450 2E1, the principal enzyme responsible for metabolizing trichloroethylene to chloral hydrate, only in males, which resulted in similar expression levels in both sexes after the treatment. Trichloroethylene influenced neither the level of catalase, an enzyme involved in the reduction of oxidative stress, nor aldehyde dehydrogenase, the main enzyme catalyzing the conversion to trichloroacetic acid. These results suggest that trichloroethylene treatment causes a male-specific PPARalpha-dependent increase in cellular oxidative stress.

Peroxisomes in permanent and provisional kidneys. Phylogenic and ontogenic considerations.

Peroxisomes in three forms of vertebrate kidney (pronephros, mesonephros, and metanephros), as permanent or provisional kidney, are summarized concerning their ultrastructure and developmental changes. Because the peroxisome is known to be diverse in mammalian metanephros, and species difference is its distinctive feature among cell organelles, information should be obtained on each kidney of each species. The ultrastructural and biochemical features of peroxisomes have at least been partly delineated in the metanephros and mesonephros, but nothing is known about the pronephros. Ultrastructural studies of the metanephric peroxisomes are present in mammals, birds, and reptiles, but information on their development is restricted to mammals and birds. As for the mesonephric peroxisomes, both ultrastructural and developmental data have been accumulating on mammals and amphibians, and ultrastructural information is present on fishes, but not on birds and reptiles. At present, studies on peroxisomes of provisional kidney have been restricted to mammalian mesonephros. The common features of renal peroxisomes previously examined are that they are spherical cell organelles with a single limiting membrane in ultrastructure, and are positive for catalase. Information on the ultrastructure and enzymes is not sufficient at present for comparing the ontogenesis of renal peroxisomes with their phylogenesis.

Three-dimensional structure of the micro-blood vessels in thyroid tumors analyzed by immunohistochemistry coupled with image analysis.

The structure of micro-blood vessels, one of the most important factors influencing the tumor growth and tumor metastasis among histological types of thyroid malignancy, was analyzed immunochemically by staining tissues for platelet endothelial cell adhesion molecule-1 (PECAM-1). Human thyroid tumor tissue obtained at surgery, consisting of 18 cases of papillary carcinoma, 9 cases of follicular carcinoma, and 9 cases of anaplastic carcinoma were fixed in formalin solution, and paraffin sections were made. They were stained for PECAM-1 using the avidin-biotin complex (ABC) technique. The volume of the blood vessels and their three-dimensional (3D) structure were analyzed using an image analyzer. The volume ratios of blood vessels in thyroid tissues were: normal tissues, 1.10%; papillary carcinoma, 3.01%; follicular carcinoma, 8.13%; and anaplastic carcinoma, 0.91%. Ratios in malignant tumors were larger than in normal tissues, except for anaplastic carcinoma. The typical 3D structure of micro-blood vessels was histopathologically varied: branching tree-like blood vessels in papillary carcinomas; vessels of varied diameter surrounding follicle structure in follicular carcinomas; and simple and immature vessels in anaplastic carcinomas. The volume and 3D structure of micro-blood vessels in thyroid malignant tumors differed from those in normal tissues, and varied according to histological classification.

Quantification of protein A-gold staining for peroxisomal enzymes by confocal laser scanning microscopy.

The protein A-gold technique has been widely applied for visual localization and quantification of various antigens by electron microscopy. Observation of specimens stained by the protein A-gold technique with conventional light microscopy is difficult because of insufficient sensitivity of the staining. Light microscopic visualization and quantification of the reaction products were attempted employing a confocal laser scanning microscope (CLSM). Liver tissues of normal and peroxisome proliferator-treated rats were fixed and embedded in Lowicryl K4M resin. Ultrathin and thin sections were stained for catalase and a peroxisome-specific beta-oxidation enzyme by the protein A-gold technique. Ultrathin sections were observed by electron microscopy and the labeling density for each enzyme was analyzed with an image analyzer. Thin sections were observed with a CLSM in the reflection mode and the intensity of the light reflection was analyzed under the same conditions for all specimens. A comparison of these two observation procedures was also attempted using liver tissues stained with various concentrations of the antibody for catalase. The intensity of the reflection for each, as observed by CLSM, correlated well with the labeling density observed by electron microscopy. CLSM made it possible to quantify and to directly observe protein A-gold staining at the light microscopic level.(J Histochem Cytochem 47:1343-1349, 1999)

Rat peroxisome proliferator-activated receptors and brown adipose tissue function during cold acclimatization.

Brown adipose tissue (BAT) hyperplasia is a fundamental physiological response to cold; it involves an acute phase of mitotic cell growth followed by a prolonged differentiation phase. Peroxisome proliferator-activated receptors (PPARs) are key regulators of fatty acid metabolism and adipocyte differentiation and may therefore mediate important metabolic changes during non-shivering thermogenesis. In the present study we have investigated PPAR mRNA expression in relation to peroxisome proliferation in rat BAT during cold acclimatization. By immunoelectron microscopy we show that the number of peroxisomes per cytoplasmic volume and acyl-CoA oxidase immunolabeling density remained constant (thus increasing in parallel with tissue mass and cell number) during the initial proliferative phase and the acute thermogenic response but increased after 14 days of cold exposure, correlating with terminal differentiation of BAT. A pronounced decrease in BAT PPARalpha and PPARgamma mRNA levels was found within hours of exposure to cold, which was reversed after 14 days, suggesting a role for either or both of these subtypes in the proliferation and induction of peroxisomes and peroxisomal beta-oxidation enzymes. In contrast, PPARdelta mRNA levels increased progressively during cold exposure. Transactivation assays in HIB 1B and HEK-293 cells demonstrated an adrenergic stimulation of peroxisome proliferator response element reporter activity via PPAR, establishing a role for these nuclear receptors in hormonal regulation of gene transcription in BAT.

Immunoelectron microscopy of peroxisomes employing the antibody for the SKL sequence PTS1 C-terminus common to peroxisomal enzymes.

Immunohistochemistry employing a new hapten antibody that detects the SKL sequence and its variants of the PTS1 C-terminus of peroxisomal enzymes was attempted to visualize peroxisomes across species. Rabbits were immunized with the SKL sequence coupled with KLH, between which an arm molecule was interposed. IgG fractions of antisera were affinity-purified against the hapten and employed for immunochemical analyses and immunoelectron microscopy. The specificity of the antibody was examined by immunoblot analyses for various purified enzymes of rat liver peroxisomes and by dot-blot analyses inhibited by SKL peptide and its variants. Various animal and plant tissues were subjected to immunoelectron microscopy with the protein A-gold technique. The antibody reacted with various enzymes in the peroxisome with the SKL motif. The affinity of the antibody for tripeptides, which varied depending on their structures, was higher for SKL than for its variants. Hepatic and renal peroxisomes of vertebrates, peroxisomes in the fat body of an insect, and the cotyledon of a plant were visualized by immunoelectron microscopy. Immunohistochemistry employing this SKL antibody may provide specific staining that can detect peroxisomes across different species.

Peroxisomal and mitochondrial fatty acid beta-oxidation in mice nullizygous for both peroxisome proliferator-activated receptor alpha and peroxisomal fatty acyl-CoA oxidase. Genotype correlation with fatty liver phenotype.

Fatty acid beta-oxidation occurs in both mitochondria and peroxisomes. Long chain fatty acids are also metabolized by the cytochrome P450 CYP4A omega-oxidation enzymes to toxic dicarboxylic acids (DCAs) that serve as substrates for peroxisomal beta-oxidation. Synthetic peroxisome proliferators interact with peroxisome proliferator activated receptor alpha (PPARalpha) to transcriptionally activate genes that participate in peroxisomal, microsomal, and mitochondrial fatty acid oxidation. Mice lacking PPARalpha (PPARalpha-/-) fail to respond to the inductive effects of peroxisome proliferators, whereas those lacking fatty acyl-CoA oxidase (AOX-/-), the first enzyme of the peroxisomal beta-oxidation system, exhibit extensive microvesicular steatohepatitis, leading to hepatocellular regeneration and massive peroxisome proliferation, implying sustained activation of PPARalpha by natural ligands. We now report that mice nullizygous for both PPARalpha and AOX (PPARalpha-/- AOX-/-) failed to exhibit spontaneous peroxisome proliferation and induction of PPARalpha-regulated genes by biological ligands unmetabolized in the absence of AOX. In AOX-/- mice, the hyperactivity of PPARalpha enhances the severity of steatosis by inducing CYP4A family proteins that generate DCAs and since they are not metabolized in the absence of peroxisomal beta-oxidation, they damage mitochondria leading to steatosis. Blunting of microvesicular steatosis, which is restricted to few liver cells in periportal regions in PPARalpha-/- AOX-/- mice, suggests a role for PPARalpha-induced genes, especially members of CYP4A family, in determining the severity of steatosis in livers with defective peroxisomal beta-oxidation. In age-matched PPARalpha-/- mice, a decrease in constitutive mitochondrial beta-oxidation with intact constitutive peroxisomal beta-oxidation system contributes to large droplet fatty change that is restricted to centrilobular hepatocytes. These data define a critical role for both PPARalpha and AOX in hepatic lipid metabolism and in the pathogenesis of specific fatty liver phenotype.

Prenatal diagnosis of peroxisomal D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein deficiency.

The prenatal diagnosis of peroxisomal D-3-hydroxyacyl-coenzyme A (CoA) dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (D-BP) deficiency was performed by peroxisomal beta-oxidation assay, indirect immunofluorescence staining, immunoblot analysis, and gene analysis of cultured amniocytes obtained from a fetus at 16 weeks' gestational age. beta-Oxidation activity, measured by [1-14C] lignoceric acid oxidation, was markedly decreased compared with the controls. Large peroxisomes were readily identified by immunofluorescence staining with anti-human catalase, as was found in the reported patients. Immunoreactive D-BP material was absent on immunoblot analysis and immunofluorescence staining with anti-human D-BP. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed the presence of the same 237-bp deletion in the cDNA as that detected in a sibling (the proband). The autopsied fetus showed the characteristic facial appearance and D-BP was deficient on immunoblot and immunohistopathological studies of the fetal tissues. No neuronal migration disorder was identified. This seems to be the first prenatal diagnosis of D-BP deficiency.

Absence of spontaneous peroxisome proliferation in enoyl-CoA Hydratase/L-3-hydroxyacyl-CoA dehydrogenase-deficient mouse liver. Further support for the role of fatty acyl CoA oxidase in PPARalpha ligand metabolism.

Peroxisomes contain a classical L-hydroxy-specific peroxisome proliferator-inducible beta-oxidation system and also a second noninducible D-hydroxy-specific beta-oxidation system. We previously generated mice lacking fatty acyl-CoA oxidase (AOX), the first enzyme of the L-hydroxy-specific classical beta-oxidation system; these AOX-/- mice exhibited sustained activation of peroxisome proliferator-activated receptor alpha (PPARalpha), resulting in profound spontaneous peroxisome proliferation in liver cells. These observations implied that AOX is responsible for the metabolic degradation of PPARalpha ligands. In this study, the function of enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase (L-PBE), the second enzyme of this peroxisomal beta-oxidation system, was investigated by disrupting its gene. Mutant mice (L-PBE-/-) were viable and fertile and exhibited no detectable gross phenotypic defects. L-PBE-/- mice showed no hepatic steatosis and manifested no spontaneous peroxisome proliferation, unlike that encountered in livers of mice deficient in AOX. These results indicate that disruption of classical peroxisomal fatty acid beta-oxidation system distal to AOX step does not interfere with the inactivation of endogenous ligands of PPARalpha, further confirming that the AOX gene is indispensable for the physiological regulation of this receptor. The absence of appreciable changes in lipid metabolism also indicates that enoyl-CoAs, generated in the classical system in L-PBE-/- mice are diverted to D-hydroxy-specific system for metabolism by D-PBE. When challenged with a peroxisome proliferator, L-PBE-/- mice showed increases in the levels of hepatic mRNAs and proteins that are regulated by PPARalpha except for appreciable blunting of peroxisome proliferative response as compared with that observed in hepatocytes of wild type mice similarly treated. This blunting of peroxisome proliferative response is attributed to the absence of L-PBE protein in L-PBE-/- mouse liver, because all other proteins are induced essentially to the same extent in both wild type and L-PBE-/- mice.

Presence of molecular chaperones, heat shock cognate (Hsc) 70 and heat shock proteins (Hsp) 40, in the postsynaptic structures of rat brain.

The synaptic localization of molecular chaperones, heat shock cognate protein 70 (Hsc70) and Hsp40, was investigated immunohistochemically in the normal rat brain. Postsynaptic density (PSD) fractions contained a constitutive form of HSP70, heat shock cognate protein 70 (Hsc70 or p73) but not inducible form of HSP70 (p72). The immunoreactivities of Hsc70 (p73) were distributed throughout the rat brain, in neuronal somata, dendrites and axons. Their immunoreactivity in neurons was localized in the cytoplasmic matrix, dendrites, and spines at the electron microscopic level. Presynaptic terminals, but less frequently than postsynaptic staining, were also reactive. Postsynaptic areas immediately beneath the synaptic contact or PSDs were immunoreactive for Hsc70. The Hsp40 was highly concentrated in PSD fractions. The staining of Hsp40 immunoreactivity was punctate and distributed widely in the brain. Hsp40 immunoreactivity was localized in dendritic spines, especially in the subsynaptic web, with weak staining of PSDs at the electron microscopic level. Double immunofluorescent staining and confocal microscopy revealed that Hsc70 and Hsp40 were co-localized on somata and neuronal processes of cultured cerebral neurons, on which synaptophysin immunoreactive spots were scattered. These results suggest that Hsp40 and Hsc70 are co-localized at postsynaptic sites and postsynaptic chaperone activity may be mediated by these two heat shock proteins.

Cloning and expression of the mouse deoxyuridine triphosphate nucleotidohydrolase gene: differs from the rat enzyme in that it lacks nuclear receptor interacting LXXLL motif.

We have previously reported the cloning of rat deoxyuridine triphosphate nucleotidohydrolase (dUTPase) cDNA and demonstrated that the full-length protein as well as the N-terminal 62-amino acid peptide interacts with peroxisome proliferator-activated receptor alpha (PPARalpha). We now report the cloning of mouse dUTPase cDNA and show that it contains a 162-amino acid open reading frame, encoding a protein with a predicted Mr of 17,400 and differs from rat cDNA, which contains additional 43 amino acids at the N-terminal end. Unlike rat dUTPase, mouse dUTPase failed to bind PPARalpha. An evaluation of 205 amino acid containing rat dUTPase cDNA revealed that the N-terminal 43 extra amino acid segment contains an LXXLL signature motif, considered necessary and sufficient for the binding of several cofactors with nuclear receptors, and its absence in murine dUTPase possibly accounts for the differential binding of these enzymes to PPARalpha. In situ hybridization and immunohistochemical studies revealed that, in the adult mouse, dUTPase is expressed at high levels in proliferating cells of colonic mucosa, and of germinal epithelium in testis. At 9.5-day mouse embryonic development, dUTPase expression is predominantly in developing neural epithelium, and hepatic primordium, and in later developmental stages (11.5-, 13.5-, and 15.5-day embryo), the expression began to be localized to the liver, kidney, gut epithelium, thymus, granular layer of the cerebellum, and olfactory epithelium. We also show that the murine dUTPase gene comprises 6 exons and the 5'-flanking region of -1479 to -27, which exhibited high promoter activity, contains a typical TATA box and multiple cis-elements such as Sp-1, AP2, AP3, AP4, Ker1, RREB, and CREB binding sites. These observations suggest the existence of variants of dUTPase, some of which may influence nuclear receptor function during development and differentiation, in addition to catalyzing the hydrolysis of dUTP to dUMP.

Occurrence of a transcription factor, cAMP response element-binding protein (CREB), in the postsynaptic sites of the brain.

The postsynaptic density (PSD) fraction prepared from the rat forebrain contained a transcription factor, cAMP response element-binding protein (CREB). The occurrence of CREB in the PSD was confirmed by immunoelectron microscopic examination. CREB in the PSD fraction was phosphorylated both by protein kinase A and Ca2+/calmodulin-dependent protein kinase II (CaMKII) endogenous to the fraction, and dissociated from the PSD after phosphorylation, especially under CaMKII-activated conditions. The fraction containing CREB that was released from PSD after phosphorylation possessed cAMP response element (CRE)-binding activity. Thus, PSD anchors functionally active CREB. These results suggest that CREB anchored to the PSD is liberated by phosphorylation upon specific synaptic stimulation, translocates into the nucleus, and then triggers synaptic activity-dependent changes in gene expression.