Is fish oil (n-3 fatty acids) effective for the maintenance of remission in Crohn's disease?
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Crohn Disease↗
Biomedical subjects
A Nakazawa
Publications and source records attributed to A Nakazawa.
Regulatory effect of interleukin-4 and interleukin-13 on colon cancer cell adhesion.
To assess the role of interleukin-4 (IL-4) and interleukin-13 (IL-13) in colon cancer cell-cell adhesion, we investigated the effect of both cytokines in human colon cancer cell line, colo205 cell-cell adhesion. IL-4 receptor was expressed on the cell surface of colo205, and recombinant IL-4 inhibited colo205 cell-cell adhesion in a dose-dependent fashion without inhibiting cell proliferation. Flow cytometric analysis revealed that monoclonal antibodies (mAbs) directed against E-cadherin and carcinoembryonic antigen (CEA) inhibited colo205 cell-cell adhesion and IL-4 significantly inhibited the expression of E-cadherin and CEA. IL-13 also inhibited colo205 cell-cell adhesion. These results indicated that IL-4 and IL-13 inhibited colon cancer cell-cell adhesion by down-regulation of E-cadherin and CEA molecules. We then investigated the expression of both cytokines from freshly isolated colon cancer tumour-infiltrating lymphocytes (TILs). With reverse transcription-polymerase chain reaction and flow cytometric analysis, we demonstrated that colon TILs expressed IL-4 and IL-13 mRNA and protein. These results suggest that Th 2 type cytokines IL-4 and IL-13 locally-produced from TILs may regulate colon cancer adhesion by down-regulation of adhesion molecules.
Cadherins↗
Long-term effect of antihypertensive therapy with calcium antagonist or angiotensin converting enzyme inhibitor on serum nitrite/nitrate levels in human essential hypertension.
Endothelial function is impaired in hypertension. In the present study the effects of long-term antihypertensive therapy on endothelial production of nitric oxide (NO) were investigated. Fifteen untreated mild to moderate essential hypertensive patients and 13 normotensive subjects were enrolled in this study. Blood pressure, heart rate, lipid profiles, cyclic guanosine 3', 5'-monophosphate (cGMP) and nitrite/nitrate (NOx), which are stable metabolites of NO, were measured. The hypertensive patients were treated with a calcium antagonist, benidipine (CAS 91559-74-5) (Ca group: n = 8) or an angiotensin converting enzyme inhibitor, trandolapril (CAS 87679-37-6) (ACEI group: n = 7) and 12 weeks after the treatment the same examinations were performed. NOx and cGMP levels in hypertensive patients were significantly lower than those in normotensive subjects (32.3 +/- 4.1 versus 49.0 +/- 6.5 mumol/l and 2.16 +/- 0.39 versus 3.39 +/- 0.42 pmol/ml, respectively). Both antihypertensive agents decreased the elevated blood pressure (mean blood pressure; 120 +/- 3 to 99 +/- 3 mmHg in Ca group and 117 +/- 4 to 104 +/- 4 mmHg in ACEI group) and normalized the decreased NOx and cGMP levels (29.1 +/- 6.2 to 46.2 +/- 8.6 mumol/l and 1.96 +/- 0.37 to 3.20 +/- 0.71 pmol/ml in Ca group, 36.0 +/- 5.3 to 54.7 +/- 6.9 mumol/l and 2.45 +/- 0.52 to 2.87 +/- 0.43 pmol/ml in ACEI group, respectively). Either benidipine or trandolapril improves the endothelial function and increases the impaired basal release of NO in hypertension. This suggests the beneficial effects of the drugs on protection against the vascular complications in hypertension.
Adult↗
Comparison of outer membrane protein genes omp and pmp in the whole genome sequences of Chlamydia pneumoniae isolates from Japan and the United States.
Chlamydia pneumoniae is a widespread pathogen of the respiratory tract that is also associated with atherosclerosis. The whole genome sequence was determined for a Japanese isolate, C. pneumoniae strain J138. The sequence predicted a variety of genes encoding outer membrane proteins (OMPs) including ompA and porB, another 10 predicted omp genes, and 27 pmp genes. All were detected in the whole genome sequence of strain CWL029, a strain isolated and sequenced in the United States. A comparative study of the OMPs of the two strains revealed a nucleotide sequence identity of 89.6%-100% (deduced amino acid sequence identity, 71.1%-100%). The overall genomic organization and location of genes are identical in both strains. Thus, a few unique sequences of the OMPs may be essential for specific attributes that define the differential biology of two C. pneumoniae strains.
Bacterial Outer Membrane Proteins↗
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.
Animals↗
[A Japanese case of idiopathic hemochromatosis with analysis of HFE gene mutations and a review of literature on HLA phenotypes in the Japanese cases].
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Asian People↗
Characterization of the 5'-flanking region of the gene encoding bovine adenylate kinase isozyme 3.
We have characterized the 5'-flanking region of the gene encoding bovine adenylate kinase isozyme 3 (AK3). S1 mapping analysis revealed multiple transcription start points in the bovine AK3 gene. The promoter activities were tested in HeLa cells using the chloramphenicol acetyltransferase (CAT) gene as a reporter. The CAT analysis showed that the basal promoter sequence was located within the region from -189 to +228. In the presence of short DNA fragments of the 5'-flanking region as competitors, the transcriptional activity of the bovine AK3 promoter changed depending on the fragments used. The results identified the basal regulatory elements in the proximal promoter region.
Adenylate Kinase↗
Cloning and functional characterization of the promoter region of the gene encoding human adenylate kinase isozyme 3.
The 5'-flanking region of the gene encoding human adenylate kinase isozyme 3 was isolated and compared with that of the bovine AK3 gene previously characterized. Four conserved DNA sequences (elements-a, -b, -c, and -d) were found in both the regions. The promoter activities were analyzed in HeLa cells using promoter-CAT reporter constructs. The proximal promoter region (-217 to +261), which contains three of four conserved elements, gave a maximum promoter activity. In a series of electrophoretic mobility-shift assays, DNA fragments and double-stranded oligodeoxyribonucleotides containing sequences of the four conserved elements interacted with nuclear extracts of HeLa cells. The a-element contained the W-element, while the d-element, which had a high G + C content, was a novel regulatory cis-element distinct from the GC box. The b- and c-elements were homologous to each other and had a motif resembling downstream promoter element. Mutations of the c- and d-elements significantly reduced the promoter activity, indicating that the c- and d-elements in the AK3 promoter are crucial. These elements may also be involved in the transcriptional regulation of other TATA-less genes.
Adenylate Kinase↗
Regulation of NeuroD expression by activation of the protein kinase-C pathway in Y79 human retinoblastoma cells.
To examine the signals that regulate NeuroD expression, we analyzed the effects of activation of two major signal pathways, the protein kinase A (PKA) pathway and the protein kinase C (PKC) pathway, on the expression of NeuroD in Y79human retinoblastoma cells. Activation of PKC resulted in marked induction of NeuroD mRNA and NeuroD protein. NeuroD mRNA induction was inhibited by calphostin C, an inhibitor of PKC. On the other hand, stimulation of PKA by forskolin had a weak suppressive effect on NeuroD mRNA expression. Induction of NeuroD expression was followed by enhancement of expression of the AK1 gene, one of the target genes of NeuroD, which encodes adenylate kinase isozyme 1, an important enzyme in the cellular adenine nucleotide homeostasis. Our results indicate that NeuroD expression is regulated, at least in part, by the PKC pathway and not by the PKA pathway.
Adenylate Kinase↗
Activation of p38 mitogen-activated protein kinase by PYK2/related adhesion focal tyrosine kinase-dependent mechanism.
The stress-activated p38 mitogen-activated protein kinase (p38 MAPK), a member of the subgroup of mammalian kinases, appears to play an important role in regulating inflammatory responses, including cytokine secretion and apoptosis. The upstream mediators that link extracellular signals with the p38 MAPK signaling pathway are currently unknown. Here we demonstrate that pp125 focal adhesion kinase-related tyrosine kinase RAFTK (also known as PYK2, CADTK) is activated specifically by methylmethane sulfonate (MMS) and hyperosmolarity but not by ultraviolet radiation, ionizing radiation, or cis-platinum. Overexpression of RAFTK leads to the activation of p38 MAPK. Furthermore, overexpression of a dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced p38 MAPK activation. MKK3 and MKK6 are known potential constituents of p38 MAPK signaling pathway, whereas SEK1 and MEK1 are upstream activators of SAPK/JNK and ERK pathways, respectively. We observe that the dominant-negative mutant of MKK3 but not of MKK6, SEK1, or MEK1 inhibits RAFTK-induced p38 MAPK activity. Furthermore, the results demonstrate that treatment of cells with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester, a membrane-permeable calcium chelator, inhibits MMS-induced activation of RAFTK and p38 MAPK. Taken together, these findings indicate that RAFTK represents a stress-sensitive mediator of the p38 MAPK signaling pathway in response to certain cytotoxic agents.
Animals↗
Overexpression of NeuroD in PC12 cells alters morphology and enhances expression of the adenylate kinase isozyme 1 gene.
NeuroD, a basic helix-loop-helix transcription factor, plays an important role in neuronal differentiation. A rat NeuroD cDNA was obtained by the aid of reverse transcription-polymerase chain reaction (RT-PCR) and ligated to an expression vector having a CMV promoter. Transfection of the NeuroD-expression plasmid into PC12 cells, a rat pheochromocytoma cell line, induced morphological changes featured by neurite-like processes and synapse-like structures without a differentiation-inducing reagent such as NGF. In the transfected cells, the overproduced NeuroD was detected by Western blot analysis, and the expression of the gene encoding mid-sized neurofilaments, a neuron-specific marker, was demonstrated by RT-PCR. Adenylate kinase isozyme 1 (AK1) is an enzyme involved in the homeostasis of energy metabolism and appears specifically in neuronal cells during differentiation. The CAT reporter assay of the 5'-flanking region of the AK1 gene suggests that NeuroD activates the AK1 expression through E-boxes in the promoter region. RT-PCR analysis indicated the enhanced level of AK1 mRNA in NeuroD-producing PC12 cells. Electrophoretic mobility shift assays demonstrated that NeuroD was able to interact with a proximal E-box element of the AK1 promoter. The results indicated that NeuroD promoted the PC12 cells to differentiate into neuron-like cells with concomitant activation of the target genes including the AK1 and the neurofilament genes.
Adenylate Kinase↗
Bcl-xL blocks activation of related adhesion focal tyrosine kinase/proline-rich tyrosine kinase 2 and stress-activated protein kinase/c-Jun N-terminal protein kinase in the cellular response to methylmethane sulfonate.
The stress-activated protein kinase/c-Jun N-terminal protein kinase (JNK) is induced in response to ionizing radiation and other DNA-damaging agents. Recent studies indicate that activation of JNK is necessary for induction of apoptosis in response to diverse agents. Here we demonstrate that methylmethane sulfonate (MMS)-induced activation of JNK is inhibited by overexpression of the anti-apoptotic protein Bcl-xL, but not by caspase inhibitors CrmA and p35. By contrast, UV-induced JNK activity is insensitive to Bcl-xL. The results demonstrate that treatment with MMS is associated with an increase in tyrosine phosphorylation of related adhesion focal tyrosine kinase (RAFTK)/proline-rich tyrosine kinase 2 (PYK2), an upstream effector of JNK and that this phosphorylation is inhibited by overexpression of Bcl-xL. Furthermore, overexpression of a dominant-negative mutant of RAFTK (RAFTK K-M) inhibits MMS-induced JNK activation. The results indicate that inhibition of RAFTK phosphorylation by MMS in Bcl-xL cells is attributed to an increase in tyrosine phosphatase activity in these cells. Hence, treatment of Bcl-xL cells with sodium vanadate, a tyrosine phosphatase inhibitor, restores MMS-induced activation of RAFTK and JNK. These findings indicate that RAFTK-dependent induction of JNK in response to MMS is sensitive to Bcl-xL, but not to CrmA and p35, by a mechanism that inhibits tyrosine phosphorylation and thereby activation of RAFTK. Taken together, these findings support a novel role for Bcl-xL that is independent of the caspase cascade.
Calcium-Calmodulin-Dependent Protein Kinases↗
Release of adenylate kinase 2 from the mitochondrial intermembrane space during apoptosis.
The release of two mitochondrial proteins, cytochrome c and apoptosis-inducing factor (AIF), into the soluble cytoplasm of cells undergoing apoptosis is well established. Using spectrophotometric determination of enzyme activity, the accumulation of adenylate kinase (AK) activity in the cytosolic fraction of apoptotic cells has also been observed recently. However, three isozymes, AK1, AK2 and AK3, have been characterized in mammalian cells and shown to be localized in the cytosol, mitochondrial intermembrane space and mitochondrial matrix, respectively, and it is unknown which one of these isozymes accumulates in the cytosol during apoptosis. We now demonstrate that in apoptotic cells only AK2 was translocated into the cytosol concomitantly with cytochrome c. The amount of AK1 in cytosol, as well as the amount of matrix-associated AK3, remained unchanged during the apoptotic process. Thus, our data suggest that only intermembrane proteins are released from mitochondria during the early phase of the apoptotic process.
Adenylate Kinase↗
Distribution and developmental changes of adenylate kinase isozymes in the rat brain: localization of adenylate kinase 1 in the olfactory bulb.
Changes in the levels of three adenylate kinase isozymes (AK1, AK2, and AK3) in the rat brain during development were investigated by immunoblot analysis. The levels of AK1 and AK3 of the whole brain increased after birth, while AK2 was detected only in the early embryonic period. In the adult rat brain, high levels of AK1 were present in the olfactory bulb. Immunohistochemical analysis showed that AK1 was found predominantly in the olfactory nerve layer and the glomerular layer. In the olfactory bulb, AK1 gene expression was enhanced in the early postnatal days, whereas it remained low in the cerebellum during the first 10 postnatal days. These results suggest that the AK isozymes are involved in neuronal maturation and regeneration. The understanding of the physiological actions of adenosine and ATP as neurotransmitters/neuromodulaters in the central nervous system has improved. ATP and adenosine receptors have been found to be widely distributed over the whole brain, although the intra- and extracellular metabolism of these compounds has not been well elucidated.
Adenylate Kinase↗
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.
Animals↗
Functional expression of costimulatory molecule CD86 on epithelial cells in the inflamed colonic mucosa.
BACKGROUND & AIMS: Costimulatory signals are essential for T-cell activation. The aim of this study was to demonstrate expression of costimulatory molecules CD86 and CD80 in human colonic epithelial cells and assess their functional roles in the activation of T cells in inflamed colonic mucosa. METHODS: CD86 and CD80 expression was assessed by immunohistochemistry of colonic mucosa, reverse-transcription polymerase chain reaction, and flow cytometric analysis of isolated colonic epithelial cells and cell lines. Costimulatory effect of CD86-expressing colonic epithelial cells on purified CD4(+) T-cell proliferation was also assessed at suboptimal phytohemagglutinin stimulation. RESULTS: CD86 and CD80 messenger RNA was detected in isolated colonic epithelial cells from normal and inflamed mucosa of patients with ulcerative colitis. Immunohistochemistry and flow cytometric analysis of colonic epithelial cells confirmed cell surface expression of CD86 protein in inflamed colonic mucosa. Cell surface expression of CD86 protein was increased in the colonic epithelial cell line HT29-18-N2 after interferon gamma stimulation. Purified CD4(+) T cells proliferated in response to suboptimal phytohemagglutinin costimulated with interferon gamma-stimulated HT29-18-N2, and these proliferative responses were efficiently inhibited by the addition of anti-CD86 monoclonal antibody. Costimulatory effect of CD86-expressing colonic epithelial cells isolated from inflamed mucosa of ulcerative colitis was also demonstrated at suboptimal phytohemagglutinin stimulation in CD4(+) T cells. CONCLUSIONS: Colonic epithelial cells may act as antigen-presenting cells, and contribute to the activation of T cells through costimulatory molecule CD86 expression in inflamed colonic mucosa.
Antigen-Presenting Cells↗
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.
Animals↗
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)
Animals↗