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Publications and source records attributed to S Maeda.
Sequences of the folP1, rpoB, and gyrA genes were analyzed for 88 isolates of Mycobacterium leprae from leprosy patients in Japan, Haiti, Indonesia, Pakistan, and the Philippines. Thirteen isolates (14.8%) showed representative mutations in more than two genes, suggesting the emergence of multidrug-resistant M. leprae.
Nucleic acid amplification was performed for five loci in the cag pathogenicity island (PAI) of Helicobacter pylori (comprising cagA, the cagA promoter region, cagE, cagT, and the left end of cagII [LEC]), and gastric inflammation in patients was evaluated. Of 204 H. pylori isolates from Japanese patients (53 with peptic ulcer, 55 with gastric cancer, and 96 with chronic gastritis), 197 (96.6%) were positive for all five loci. Two isolates (1%) were negative for all five loci, and five isolates (2.4%) were positive for only cagA and LEC. These latter seven isolates were all from patients with mild chronic gastritis. Neutrophil infiltration in gastric mucosa was significantly milder in patients infected with partially or totally deleted-PAI strains than in those with intact-PAI strains. The cagE gene was a more accurate marker of an intact cag PAI than the cagA gene, and cagE seemed to be more useful in discriminating between H. pylori strains causing different rates of disease progression.
Helicobacter pylori induces cellular proliferation in host cells, but the mechanism remains unclear. Thus, we examined the effect of H. pylori on cyclin D1, an important regulator of the cell cycle, especially in relation to intracellular signaling pathways. In a Northern blot analysis, cyclin D1 transcription in gastric cancer (AGS) cells was enhanced by coculture with H. pylori strain TN2 in a time-dependent and multiplicity-of-infection-dependent manner. An isogenic mutant form of vacA also increased cyclin D1 transcription, but mutant forms of cagE or the entire cag pathogenicity island did not enhance cyclin D1 transcription. These effects were confirmed with a luciferase assay of the cyclin D1 promoter (pD1luc). Cyclin D1 promoter activation by H. pylori was inhibited by MEK inhibitors (U0126 and PD98059), indicating that the mitogen-activated protein kinase pathway may be involved in intracellular signal transduction. In contrast, transfection of a reporter plasmid having any point mutations of the NF-kappaB binding sites in the promoter (pD1-kappaB1M, pD1-kappaB2M, or pD1-kappaB1/2M) or cotransfection of dominant negative IkappaBalpha did not affect cyclin D1 activation by H. pylori. In conclusion, H. pylori activates cyclin D1 through the mitogen-activated protein kinase pathway and not through NF-kappaB activation in AGS cells. This activation of cyclin D1 is partly dependent on the cag pathogenicity island but not on vacA.
The cmpABCD operon of Synechococcus sp. strain PCC 7942, encoding a high-affinity bicarbonate transporter, is transcribed only under CO2-limited conditions. In Synechocystis sp. strain PCC 6803, the slr0040, slr0041, slr0043, and slr0044 genes, forming an operon with a putative porin gene (slr0042), were identified as the cmpA, cmpB, cmpC, and cmpD genes, respectively, on the basis of their strong similarities to the corresponding Synechococcus cmp genes and their induction under low CO2 conditions. Immediately upstream of and transcribed divergently from the Synechocystis cmp operon is a gene (sll0030) encoding a homolog of CbbR, a LysR family transcriptional regulator of the CO2 fixation operons of chemoautotrophic and purple photosynthetic bacteria. Inactivation of sll0030, but not of another closely related cbbR homolog (sll1594), abolished low CO2 induction of cmp operon expression. Gel retardation assays showed specific binding of the Sll0030 protein to the sll0030-cmpA intergenic region, suggesting that the protein activates transcription of the cmp operon by interacting with its regulatory region. A cbbR homolog similar to sll0030 and sll1594 was cloned from Synechococcus sp. strain PCC 7942 and shown to be involved in the low CO2-induced activation of the cmp operon. We hence designated the Synechocystis sll0030 gene and the Synechococcus cbbR homolog cmpR. In the mutants of the cbbR homologs, upregulation of ribulose-1,5-bisphosphate carboxylase/oxygenase operon expression by CO2 limitation was either unaffected (strain PCC 6803) or enhanced (strain PCC 7942), suggesting existence of other low CO2-responsive transcriptional regulator(s) in cyanobacteria.
BACKGROUND AND AIMS: Helicobacter pylori infection induces expression of proinflammatory cytokines such as interleukin (IL)-8 and tumour necrosis factor alpha (TNF-alpha) in gastric mucosa, and their genes have AP-1 binding sites in the promoter region. c-Fos is important for transactivation of AP-1 which has SRE in the promoter region. We conducted this study to confirm H pylori induced transactivation of these binding sites. METHODS: Transactivation of SRE and AP-1 was evaluated in human gastric cancer cells TMK1 and MKN45 by luciferase reporter assay in transient transfection. We compared the effects of coculture with four H pylori strains, a cag pathogenicity island (PAI) positive strain TN2, its isogenic vacA negative (TN2-DeltavacA) or cagE negative (TN2-DeltacagE) mutants, and a cag PAI negative clinical isolate T68. Phosphorylation of ERK1/2, JNK, and c-Jun was measured by immunoblot, induction of IL-8 secretion by ELISA, and the effects of MEK by inhibitor U0126. RESULTS: Both SRE and AP-1 were transactivated by coculture with TN2. Although TN2-DeltavacA induced comparable transactivation, TN2-DeltacagE and T68 showed decreased transactivation of SRE (65% and 51%) and AP-1 (71% and 54%, respectively, of TN2). Heat killed TN2 or indirect contact using a permeable membrane inhibited transactivation. Levels of phosphorylated ERK1/2, JNK, and c-Jun were increased by coculture with TN2. MEK inhibitor U0126 reduced TN2 induced transactivation of SRE and AP1, as well as secretion of IL-8, by 83%, 87%, and 53%, respectively, of TN2. CONCLUSIONS: Transactivation of SRE and AP-1, through ERK/MAPK and JNK/SAPK cascades, respectively, was found in gastric cancer cells cocultured with H pylori. Direct contact with viable bacteria possessing intact cag PAI is a prerequisite for the onset of intracellular signalling leading to AP-1 transactivation.
Pressure overload, such as hypertension, to the heart causes pathological cardiac hypertrophy, whereas chronic exercise causes physiological cardiac hypertrophy, which is defined as athletic heart. There are differences in cardiac properties between these two types of hypertrophy. We investigated whether mRNA expression of various cardiovascular regulating factors differs in rat hearts that are physiologically and pathologically hypertrophied, because we hypothesized that these two types of cardiac hypertrophy induce different molecular phenotypes. We used the spontaneously hypertensive rat (SHR group; 19 wk old) as a model of pathological hypertrophy and swim-trained rats (trained group; 19 wk old, swim training for 15 wk) as a model of physiological hypertrophy. We also used sedentary Wistar-Kyoto rats as the control group (19 wk old). Left ventricular mass index for body weight was significantly higher in SHR and trained groups than in the control group. Expression of brain natriuretic peptide, angiotensin-converting enzyme, and endothelin-1 mRNA in the heart was significantly higher in the SHR group than in control and trained groups. Expression of adrenomedullin mRNA in the heart was significantly lower in the trained group than in control and SHR groups. Expression of beta(1)-adrenergic receptor mRNA in the heart was significantly higher in SHR and trained groups than in the control group. Expression of beta(1)-adrenergic receptor kinase mRNA, which inhibits beta(1)-adrenergic receptor activity, in the heart was markedly higher in the SHR group than in control and trained groups. We demonstrated for the first time that the manner of mRNA expression of various cardiovascular regulating factors in the heart differs between physiological and pathological cardiac hypertrophy.
Transforming growth factor-beta(1) (TGF-beta(1)) and interleukin (IL)-10 gene expression is equivocal in normal brain and upregulated in over a dozen central and peripheral diseases/disorders. The patterns of specific expression of cytokines differ in these diseases. Published data indicate that these cytokines are produced by and act on both neurons and glial cells. Although their actions are commonly viewed as 'anti-inflammatory', they protect neurons and downregulate the responses of glial cells to diseases/disorders in the absence of inflammation. Their actions counterbalance the actions of elevated IL-1 and/or tumor necrosis factor-alpha to maintain homeostasis. Their therapeutic potential will be realized by improving our understanding of their place in neural cytokine networks.
We investigated the effect of CH-19 Sweet, a non-pungent cultivar of red pepper, on body temperature and oxygen consumption in humans. CH-19 Sweet was given to 11 healthy volunteers, and core body temperature, body surface temperature and oxygen consumption were measured. The control group ingested California-Wandar, which contained neither capsaicin nor capsiate. The core body temperature in the CH-19 Sweet group was significantly higher than that in the control group (P<0.01). The forehead temperature measured by infrared thermography in the CH-19 Sweet group was significantly higher than that in the control group. The body surface temperature was increased for about 20 min after consumption of CH-19 Sweet intake, and the neck temperature was significantly higher (P<0.001) than when the subjects consumed California-Wandar. We also measured respiratory gas by indirect calorimetry while subjects wore a face mask. A significant difference was detected in oxygen consumption between the two groups, and the value was significantly higher in the CH-19 Sweet group (P<0.03). These results suggest that CH-19 Sweet increased thermogenesis and energy consumption.
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With the development and widespread use of colonoscopy, lymphangioma of the large intestine has recently been reported frequently. This paper presents some findings from a review of 279 cases of this disease in Japan, including a typical case that we encountered. A 69-year-old female was diagnosed as having lymphangioma of the descending colon based on the findings of a barium enema and a colonoscopy, and the lesion was successfully removed by an endoscopic resection. In the published reports, the etiology of this disease is not clear yet but the age at onset range shows a tendency toward a higher incidence in comparatively older patients and the male-to-female ratio indicates a higher incidence in males. If there is no complication, endoscopic treatment seems to be the preferable procedure for this disease.
Solitary fibrous tumors (SFTs) represent a distinct neoplasm that should be included in the differential diagnosis of spindle-cell neoplasms of the soft tissue. Basic fibroblast growth factor (bFGF or FGF-2) is a mitogenic and angiogenic polypeptide produced by diverse cell types, including the cells derived from normal tissue and neoplastic lesions. In this study, the expression of bFGF, vimentin, CD 34, c-kit (or CD 117), desmin, S-100 protein, and alpha-smooth muscle actin (alpha-SMA) in SFTs, hemangiopericytomas (HPC), gastrointestinal stromal tumors (GIST), and dermatofibrosarcoma protuberans (DFSP) were evaluated to assess their usefulness in the differential diagnosis of these lesions. The expression of bFGF mRNA was also examined in SFTs by in situ hybridization (ISH) using a digoxigenin-labeled bFGF oligonucleotide probe. All the SFTs, GISTs and DFSPs exhibited strong and diffuse immunoreactivity for CD34 and vimentin, and were completely negative for desmin, S-100 protein and alpha-SMA. The HPCs were positive for vimentin, but negative for CD34. In all the SFTs, strong and diffuse nuclear immunostaining was observed with bFGF antibody, contrasting with the negative staining observed in the majority of the HPCs, GISTs, and DFSPs. The bFGF mRNA was also expressed in the SFT cells. The constitutive expression of the bFGF in the SFT widens the spectrum of available markers for these tumors, providing a useful addition to their differential diagnosis in difficult cases, and contributing to the understanding of their histogenesis and molecular pathogenesis.
Thymus and activation-regulated chemokine (TARC) is known as a functional ligand for CC chemokine receptor 4 (CCR4), which is selectively expressed on Th2 lymphocytes and induces selective migration of the cells to allergic lesions. In this study, we cloned canine TARC cDNA from canine thymus by RT-PCR with rapid amplification of cDNA ends (RACE) method. The canine TARC clone contained a full-length open reading frame encoding 99 amino acids and included four cysteine residues characteristic to CC chemokine family. The canine TARC cDNA showed 77.5%, 67.4%, and 68.5% amino acid sequence similarity with human, mouse and rat homologues, respectively. Expression of TARC mRNA was detected not only in thymus but also in spleen, lymph node, lung and heart of the various normal dog tissues examined. TARC cDNA clone obtained in this study will be useful for further investigation on allergic diseases in dogs.
Chondroitin sulfate (CS) isomers, 6-sulfate (CS6) and 4-sulfate (CS4), change their ratio to each other in cartilaginous tissues with aging. In this study, a quantitative measurement method of CS6 and CS4 was developed, using capillary electrophoresis (CE). Various buffer solutions, pH, and digestion times were studied, and the use of 0.1 M Tris-HCl at pH of 8.0 allowed the isolation of CS6 and CS4 from CS most efficiently when combined with chondrotinase ABC at a concentration of 1 mU/microg of the substrate during a 3 hr digestion period. Amounts of newly synthesized CS6 and CS4 in the intervertebral disk chondrocyte three-dimensional culture were quantified by this method after the proteoglycans were extracted by equilibrium density centrifugation.
Ossification of the posterior longitudinal ligament (OPLL) of the spine is the leading cause of myelopathy in Japan. In earlier studies, we provided genetic linkage and allelic association evidence of distinct differences in the human collagen alpha2(XI) gene (COL11A2) that might constitute inherited predisposition to OPLL. In the present study, a strong allelic association with non-OPLL (p = 0.0003) was observed with an intron 6 polymorphism [intron 6 (-4A)], in which the intron 6 (-4A) allele is more frequently observed in non-OPLL subjects than in OPLL patients. In addition, a newly identified polymorphism in exon 6 [exon 6 (+28A)] was in linkage disequilibrium with the intron 6 (-4A). The functional impact of the polymorphisms was analyzed by comparing the differences in messenger RNA (mRNA) splicing by reverse-transcription polymerase chain reaction (RT-PCR) analysis in cultured cells from the interspinous ligament and an in vitro exon trapping study. The intron 6 (-4A) allele resulted in skipping exon 6 and retaining exon 7, while the exon 6 (+28A) allele was not associated with alteration in mRNA splicing. Similar mRNA species were observed in undifferentiated osteoblast (Ob) cells and in cells from posterior longitudinal ligament of non-OPLL subjects. The region containing exons 6-8 is an acidic subdomain presumably exposed to the surface that could interact with molecules of the extracellular matrix. Accordingly, retaining exon 7 together with removal of exon 6 observed in intron 6 (-4A) could play a protective role in the ectopic ossification process because the same pattern was observed in undifferentiated Ob cells and nonossified posterior longitudinal ligament cells.
Previous studies have shown that matrix vesicles isolated from cultures of costochondral growth zone chondrocytes and treated with 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] can activate recombinant human latent transforming growth factor beta1 (rhTGF-beta1). It is unknown what enzyme or other factor in the extracellular organelles is responsible for the activation. This study tested the hypothesis that enzymes present in matrix vesicles can activate latent TGF-beta1 and that this is regulated by 1alpha,25(OH)2D3. To do this, we examined the ability of matrix vesicle extracts to activate small latent rhTGF-beta1. In addition, enzymes previously determined to be present in matrix vesicles were screened for their ability to activate small latent rhTGF-beta1. Recombinant human matrix metalloproteinase 2 (rhMMP-2; 72 kDa gelatinase), rhMMP-3 (stromelysin 1), purified human plasminogen, and purified urokinase (plasminogen activator) were each tested at varying concentrations. To assess the role of cell maturation, we used a cell culture model in which chondrocytes are derived from two distinct zones of rat costochondral cartilage, the resting zone and the growth zone. Matrix vesicles were isolated from these cultures and then tested. The results showed that extracts of matrix vesicles produced by both growth zone and resting zone chondrocytes were able to activate small latent rhTGF-beta1. The effects were dose and time dependent, with greater activity being found in extracts of matrix vesicles from the growth zone chondrocyte cultures. Only rhMMP-3 was able to activate small latent rhTGF-beta1, indicating that stromelysin-1, but not MMP-2, plasminogen, or urokinase, was involved. As observed in the extracts, the effect of rhMMP-3 was time and dose dependent. When anti-MMP-3 antibody was added to matrix vesicle extracts from both cell types, activation of small latent rhTGF-beta1 was dose-dependently blocked. Neither 1alpha,25(OH)2D3 nor 24R,25(OH)2D3 had a direct effect on activation of small latent rhTGF-beta1 by the extracts. However, when intact matrix vesicles were treated with 1alpha,25(OH)2D3, their ability to activate small latent rhTGF-beta1 was increased. Inhibition of phospholipase A2 with quinacrine blocked the 1alpha,25(OH)2D3-dependent effect. These results suggest that the ability of 1alpha,25(OH)2D3-treated matrix vesicles to activate small latent TGF-beta1 is via action of the secosteroid on the matrix vesicle membrane, not on the enzymes responsible for activating latent TGF-beta1. Because matrix vesicles isolated from growth zone chondrocytes have been shown to contain increased phospholipase A2 activity after treatment with 1alpha,25(OH)2D3, it is likely that this secosteroid promotes loss of membrane integrity through phospholipase A2-dependent formation of lysophospholipids, resulting in the release of MMP-3 into the matrix, where latent TGF-beta1 is stored. Taken together, the results of the current study show that matrix vesicles produced by growth plate chondrocytes contain MMP-3, that this enzyme is at least partially responsible for activation of small latent TGF-beta1 in the matrix, and that 1alpha,25(OH)2D3 regulates MMP release from matrix vesicles.
Recent studies indicate that 1alpha,25-dihydroxyvitamin D3 (1alpha,25[OH]2D3) and 24R,25-dihydroxyvitamim D3 (24R,25[OH]2D3) differentially regulate proliferation, differentiation, and matrix synthesis of growth plate chondrocytes. To determine whether both metabolites play the same or different roles in vivo, we used the vitamin D-deficient rat as a model. Rickets was induced and then reversed by administering a single dose of ergocalciferol, 1alpha,25(OH)2D3, or 24R,25(OH)2D3 and euthanizing the animals after 4, 24, 48, or 72 h. Growth plates were either processed for histology and histomorphometry or extracted with buffered guanidine-HCl. Neutral metalloproteinase activity in the extracts was measured by use of aggrecan-containing beads, and collagenase activity was determined by use of radioactive type I collagen. The levels of tissue inhibitor of metalloproteinases (TIMP) and plasminogen activator were also determined. The morphology of the growth plate varied as a function of treatment. While 24R,25(OH)2D3 appeared to affect cell maturation and 1alpha,25(OH)2D3 appeared to affect terminal differentiation and calcification, response to ergocalciferol was indicative of the combined responses to the individual metabolites. Enzyme activity was regulated in a differential manner. Treatment with ergocalciferol produced a rapid decline in both neutral metalloproteinase and collagenase activities that was statistically significant by 4 h. By contrast, 1alpha,25(OH)2D3 had no effect on neutral metalloproteinase activity but caused a significant decrease in both active and total collagenase activity by 4 h, while 24R,25(OH)2D3 decreased neutral metalloproteinase activity by 48 h and had no effect on collagenase activity. Ergocalciferol had no effect on TIMP levels at any time examined, whereas 1alpha,25(OH)2D3 caused an increase at 48 and 72 h and 24R,25(OH)2D3 completely blocked TIMP production at 4 and 24 h. By contrast, plasminogen activator activity by ergocalciferol was decreased at 4 h, increased by 1alpha,25(OH)2D3 at 4 and 24 h, and decreased by 24R,25(OH)2D3 at all time points examined. These in vivo results confirm our previous cell culture observations showing that growth plate chondrocytes are differentially regulated by 1alpha,25(OH)2D3 and 24R,25(OH)2D3. Moreover, they show definitively that these two vitamin D metabolites play distinct roles not only in regulating neutral metalloproteinase and collagenase activities in growth plate cartilage but in cell maturation and calcification of this tissue in vivo.
The aim of the present retrospective study was to clarify the histopathologic substrates of left ventricular myocardium with transient asynergy due to acute ischemic insult in man. Three patients who had had prolonged chest pain, new abnormal Q waves and new ST segment elevation were studied. There was no significant elevation of serum creatine phosphokinase activity in two of the three patients. Echocardiograms on admission or the next day showed severe hypokinetic or akinetic motion and thinning of the anteroseptal and apical portions of the left ventricle and regional dilatation of the same portions. Disappearance of the abnormal Q waves, ST segment elevation resolution, and early T wave inversion were observed later. Complete improvement of the echocardiographic abnormalities was confirmed after a few weeks in all patients. Manifest ischemic lesions of subendocardial scars of the anteroseptal region of the left ventricle were detected in only one of the three cases by gross examination. However, on microscopic examination, islands of necrotic myocytes were interspersed with islands of viable cells throughout the jeopardized region in one case, although the scattered necrotic foci were restricted to the subendocardium and the trabeculae. Normal myocardium and subendocardial scars were observed in the other two cases. In conclusion, left ventricular myocardium with transient asynergy. detected clinically during acute ischemic attack, consists of normal myocardium or small ischemic lesions primarily in the subendocardium.
Mechanical loads are constantly being applied to articular cartilage. However, comparatively few studies have investigated the effects of mechanical stress on the gene expression of chondrocytes. Hence, the objective of the present study was to clarify the effects of mechanical stress on cultured chondrocytes. Articular chondrocytes of rabbits were isolated and placed in minimun essential medium eagle a modification containing 10% fetal bovine serum. The chondrocytes were cultured in polylayer in polypropylene tubes. After 48 hours, centrifugal stress (2.7 MJ) was applied once every 24 hours for 28 consecutive days. The mRNA expression of insulin-like growth factor-I (IGF-I) and cartilage-specific genes (type II collagen and aggrecan) in chondrocytes, cell count, wet tissue mass and protein content were subsequently measured. The results showed that the mRNA expression of the IGF-I gene for the stress groups was significantly greater than that of the control group from days 3 to 5 of incubation, as was the mRNA expression of the type II collagen gene from days 7 to 14 of incubation. However, there were no significant differences in the mRNA expression of the aggrecan gene between the control and stress groups. The cell count for the stress groups was significantly higher from days 7 to 28 of incubation, and the wet tissue mass and protein content were significantly higher from days 14 to 28 of incubation. These findings suggest that one of the mechanisms by which mechanical stress increases the number of cultured chondrocytes and the volume of extracellular matrix is to increase IGF-I expression at the early stage of incubation.