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1.  Chronological changes in the collagen-type composition at tendon–bone interface in rabbits 
Bone & Joint Research  2012;1(9):218-224.
The purpose of this study was to evaluate chronological changes in the collagen-type composition at tendon–bone interface during tendon–bone healing and to clarify the continuity between Sharpey-like fibres and inner fibres of the tendon.
Male white rabbits were used to create an extra-articular bone–tendon graft model by grafting the extensor digitorum longus into a bone tunnel. Three rabbits were killed at two, four, eight, 12 and 26 weeks post-operatively. Elastica van Gieson staining was used to colour 5 µm coronal sections, which were examined under optical and polarised light microscopy. Immunostaining for type I, II and III collagen was also performed.
Sharpey-like fibres comprised of type III collagen in the early phase were gradually replaced by type I collagen from 12 weeks onwards, until continuity between the Sharpey-like fibres and inner fibres of the tendon was achieved by 26 weeks.
Even in rabbits, which heal faster than humans, an observation period of at least 12 to 26 weeks is required, because the collagen-type composition of the Sharpey-like fibre bone–tendon connection may have insufficient pullout strength during this period. These results suggest that caution is necessary when permitting post-operative activity in humans who have undergone intra-bone tunnel grafts.
PMCID: PMC3626213  PMID: 23610694
Tendon–bone healing; Sharpey-like fibres; Rabbits; Immunostaining; Collagen-type composition; Tendon–bone interface
5.  Characterization of a new animal model of metabolic syndrome: the DahlS.Z-Leprfa/Leprfa rat 
Nutrition & Diabetes  2011;1(1):e1-.
The DahlS.Z-Leprfa/Leprfa (DS/obese) rat strain was established from a cross between Dahl salt-sensitive rats and Zucker fatty (fa/fa) rats, the latter of which harbor a missense mutation in the leptin receptor gene (Lepr). We examined whether DS/obese rats might be a suitable animal model of metabolic syndrome in humans.
The systemic pathophysiological and metabolic characteristics of DS/obese rats were determined and compared with those of homozygous lean littermates, namely, DahlS.Z-Lepr+/Lepr+ (DS/lean) rats.
Systolic blood pressure was higher in DS/obese rats fed a normal diet than in DS/lean rats at 11 weeks of age and thereafter. The survival rate of DS/obese rats was significantly lower than that of DS/lean rats at 18 weeks. Body weight, visceral and subcutaneous fat mass, as well as heart, kidney and liver weights, were increased in DS/obese rats at 18 weeks compared with DS/lean rats. Serum low-density lipoprotein (LDL)-cholesterol, triglyceride and insulin concentrations, as well as the ratio of LDL-cholesterol to high-density lipoprotein-cholesterol levels, were increased in DS/obese rats, whereas serum glucose concentration did not differ significantly between DS/obese and DS/lean rats. Creatinine clearance was decreased and urinary protein content was increased in DS/obese rats, which also manifested lipid accumulation in the liver and elevation of serum alanine aminotransferase levels.
These results show that the phenotype of DS/obese rats is similar to that of humans with metabolic syndrome, and that these animals may thus be an appropriate model for this condition.
PMCID: PMC3302131  PMID: 23154293
metabolic syndrome; animal model; obesity; hypertension; dyslipidemia; insulin resistance
6.  Expression of granulysin in polymyositis and inclusion‐body myositis 
Granulysin, a recently defined cytolytic molecule, is expressed in cytotoxic T cells and natural killer cells in a similar way to perforin, which is reported to have a major role in the pathogenesis of polymyositis and inclusion‐body myositis (IBM).
To clarify the role of granulysin in polymyositis and IBM.
The expression of granulysin and perforin was examined by double staining with CD8, CD4 and CD56 in endomysial infiltrating cells and autoinvasive cells in muscle biopsy specimens of 17 patients with polymyositis (6 steroid resistant and 11 steroid responsive) and of 7 patients with IBM.
Similar to perforin, granulysin was expressed in CD8, CD4 or CD56 cells in patients with polymyositis and IBM. The ratio of cells double positive for granulysin and CD8 to all CD8 cells at endomysial sites was notably higher in steroid‐resistant polymyositis than in steroid‐responsive polymyositis and IBM.
Granulysin expression in CD8 cells seems to be correlated with steroid resistance in polymyositis.
PMCID: PMC2077550  PMID: 16980658
7.  Differential actions of insecticides on target sites: basis for selective toxicity 
Human & experimental toxicology  2007;26(4):361-366.
Whereas the selective toxicity of insecticides between insects and mammals has a long history of studies, it is now becoming abundantly clear that, in many cases, the differential action of insecticides on insects and mammalian target receptor sites is an important factor. In this paper, we first introduce the mechanism of action and the selective toxicity of pyrethroids as a prototype of study. Then, a more detailed account is given for fipronil, based primarily on our recent studies. Pyrethroids keep the sodium channels open for a prolonged period of time, causing elevation of the depolarizing after-potential. Once the after-potential reaches the threshold for excitation, repetitive after-discharges are produced, resulting in hyperexcitation of intoxicated animals. Only about 1% of sodium channels needs to be modified to produce hyperexcitation, indicating a high degree of toxicity amplification from sodium channels to animals. Pyrethroids were > 1000-fold more potent on cockroach sodium channels than rat sodium channels, and this forms the most significant factor to explain the selective toxicity of pyrethroids in insects over mammals. Fipronil, a phenylpyrazole, is known to act on the γ-aminobutyric acid receptor to block the chloride channel. It is effective against certain species of insects that have become resistant to most insecticides, including those acting on the γ-aminobutyric acid receptor, and is much more toxic to insects than to mammals. Recently, fipronil has been found to block glutamate-activated chloride channels in cockroach neurons in a potent manner. Since mammals are devoid of this type of chloride channel, fipronil block of the glutamate-activated chloride channel is deemed responsible, at least partially, for the higher selective toxicity to insects over mammals and for the lack of cross-resistance.
PMCID: PMC2677724  PMID: 17615118
fipronil; GABA receptor; glutamate-activated chloride channel; pyrethroid; selective toxicity; sodium channel
8.  Monocyte chemoattractant protein 1 and macrophage cyclooxygenase 2 expression in colonic adenoma 
Gut  2006;55(1):54-61.
Background and aims
Cyclooxygenase 2 (COX‐2) expression in subepithelial macrophages of colorectal adenoma has been suggested as the first in a series of steps leading to colorectal tumorigenesis. We tested the hypothesis that chemokines released from human colorectal adenoma epithelium might be involved in COX‐2 expression in macrophages of the lamina propria.
Endoscopic samples of sporadic colorectal adenomas were tested by enzyme linked immunosorbent assay for chemokines involved in macrophage chemotaxis. Localisation of adenoma macrophage chemoattractant protein 1 (MCP‐1) and COX‐2 were determined by immunohistochemistry. The effects of MCP‐1, in the presence or absence of celecoxib, on COX‐2 expression, and prostaglandin (PG) E2 and vascular endothelial growth factor (VEGF) release, were examined in human macrophages isolated from peripheral blood.
MCP‐1 levels were markedly higher in adenoma with mild‐moderate dysplasia (129.7 (19.9) pg/mg protein) and severe dysplasia (227.9 (35.4) pg/mg protein) than in normal colonic mucosa (55.8 (4.2) pg/mg protein). Other chemokine levels, macrophage inflammatory proteins (MIP)‐1α and MIP‐1β, and the chemokine regulated on activation of normal T cell expressed and secreted (RANTES) did not vary significantly between adenoma and normal mucosa. MCP‐1 levels in both adenoma and normal colonic mucosa increased significantly three hours after tissue cultivation in vitro. MCP‐1 immunoreactivity was restricted to the adenoma epithelium, with no reactivity seen in adjacent normal epithelial cells. MCP‐1 stimulated COX‐2 expression and PGE2 and VEGF release in human macrophages. Celecoxib, a selective COX‐2 inhibitor, inhibited MCP‐1‐induced PGE2 and VEGF release in macrophages. Addition of exogenous PGE2 reversed this inhibitory effect on VEGF release, suggesting that MCP‐1 in adenoma epithelial cells might be involved in COX‐2 expression and subsequent macrophage activation.
MCP‐1 in colorectal adenoma epithelial cells might be involved in macrophage migration and COX‐2 expression, leading to the subsequent development of colonic adenoma.
PMCID: PMC1856393  PMID: 16085694
cyclooxygenase; macrophage chemoattractant protein; adenoma; macrophage
9.  MUC4 expression is a novel prognostic factor in patients with invasive ductal carcinoma of the pancreas 
Journal of Clinical Pathology  2005;58(8):845-852.
Background: Many patients with invasive ductal carcinoma of the pancreas (IDC) have a poor outcome. MUC4 expression has been implicated as a marker for diagnosis and progression of IDC, but there are no studies of the relation between MUC4 expression and patient prognosis in IDC.
Aims: To investigate the prognostic significance of MUC4 expression in IDC.
Methods: The expression profiles of MUC4, ErbB2, p27, and MUC1 were investigated in IDC tissues from 135 patients by means of immunohistochemistry.
Results: MUC4 was expressed in 43 of the 135 patients with IDC (31.9%). The survival of 21 patients with high MUC4 expression (>20% of neoplastic cells stained) was significantly worse than that of the 114 patients with low MUC4 expression (<20% of neoplastic cells stained) (p  =  0.0043). Univariate analysis showed that high MUC4 expression (p  =  0.0061), large primary tumour status (>T2) (p  =  0.0436), distant metastasis (p  =  0.0383), lymphatic invasion (p  =  0.0243), and surgical margins (p  =  0.0333) were significant risk factors affecting the outcome of patients with IDC. Backward stepwise multivariate analysis showed that MUC4 expression (p  =  0.0121), lymph node metastasis (p  =  0.0245), and lymphatic invasion (p  =  0.0239) were significant independent risk factors. ErbB2, p27, and MUC1 were not independent risk factors.
Conclusions: This study shows that MUC4 expression in IDC is a new independent factor for poor prognosis and predicts the outcome of patients with IDC.
PMCID: PMC1770880  PMID: 16049287
pancreatic cancer; mucin; immunohistochemistry; cumulative survival rate; multivariate analysis
10.  Doctors' and nurses' attitudes towards and experiences of voluntary euthanasia: survey of members of the Japanese Association of Palliative Medicine 
Journal of Medical Ethics  2001;27(5):324-330.
Objective—To demonstrate Japanese doctors' and nurses' attitudes towards and practices of voluntary euthanasia (VE) and to compare their attitudes and practices in this regard.
Design—Postal survey, conducted between October and December 1999, using a self-administered questionnaire.
Participants—All doctor members and nurse members of the Japanese Association of Palliative Medicine.
Main outcome measure—Doctors' and nurses' attitude towards and practices of VE.
Results—We received 366 completed questionnaires from 642 doctors surveyed (response rate, 58%) and 145 from 217 nurses surveyed (68%). A total of 54% (95% confidence interval (CI): 49-59) of the responding doctors and 53% (CI: 45-61) of the responding nurses had been asked by patients to hasten death, of whom 5% (CI: 2-8) of the former and none of the latter had taken active steps to bring about death. Although 88% (CI: 83-92) of the doctors and 85% (CI: 77-93) of the nurses answered that a patient's request to hasten death can sometimes be rational, only 33% (CI: 28-38) and 23% (CI: 16-30) respectively regarded VE as ethically right and 22% (CI: 18-36) and 15% (CI: 8-20) respectively would practise VE if it were legal. Logistic regression model analysis showed that the respondents' profession was not a statistically independent factor predicting his or her response to any question regarding attitudes towards VE.
Conclusions—A minority of responding doctors and nurses thought VE was ethically or legally acceptable. There seems no significant difference in attitudes towards VE between the doctors and nurses. However, only doctors had practised VE.
Key Words: Euthanasia • Japan • doctors • nurses • palliative care
PMCID: PMC1733435  PMID: 11579190
11.  Stenting of Extracranial Carotid Artery Stenosis 
Interventional Neuroradiology  2004;9(Suppl 1):137-141.
The purpose of this study is to evaluate our cases of cervical internal carotid artery stenosis for safty stenting. We investigate the preoperative internal carotid artery stenosis using by integrated backscatter (IBS) method of ultra sonography, comparing with the thirty five surgical specimens as to their nature, histological structure, thickness of fibrous cap. We choose the protection method according to plaque structure, and placed Easy-Wall stent or Smart stent after prePTA. We added post PTA according to the extent of expansion and IVUS findings. Calibrated IBS = IBS value (ROI) /intinal IBS value of ‘bleeding’, ‘lipiď, ‘thrombus’, fiber, ‘hyalinization’ were -27.5, -22.5, -15.2, -11.1, +2.1. That of the thin fibrous cap were -10.9*, that of thic fibrous cap were -2.4 (*p < 0.001). There was a good coleration between the extent of expansion and expected histological findings. All conplications were two cases of small cerebral infarction and a case of bleeding from the complicated lung cancer. The protection at prePTA lead to no complications in case of acute cerebral infarctions. It is very important to check the histological specimen carefully for safty stenting.
PMCID: PMC3553469  PMID: 20591243
stenting, carotid stenosis, evaluation
12.  Dexamethasone and nitric oxide synthase gene expression in brain. 
Systemic administration of lipopolysaccharide (LPS), which causes endotoxemia and systemic inflammation, has been reported to induce expression of the gene for type II inducible nitric oxide synthase (iNOS) in peripheral organs. This study was carried out to examine whether intraperitoneally injected LPS elicits the expression of iNOS messenger ribonucleic acid (mRNA) in the rat brain. We also investigated whether intraperitoneal treatment with dexamethasone (DEX) prevents this induction. To determine levels of iNOS mRNA, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method was employed. Treatment with LPS induced the expression of iNOS mRNA in various brain regions, accounting for approximately 1 x 10(5) to 4 x 10(5) molecules per micrograms of poly A+ RNA, and these inductions were markedly suppressed by DEX. The results suggest that, during systemic inflammation, iNOS mRNA induction occurs in brain through a DEX-sensitive mechanism.
PMCID: PMC1188832  PMID: 9074304
13.  HSF4, a new member of the human heat shock factor family which lacks properties of a transcriptional activator. 
Molecular and Cellular Biology  1997;17(1):469-481.
Heat shock transcription factors (HSFs) mediate the inducible transcriptional response of genes that encode heat shock proteins and molecular chaperones. In vertebrates, three related HSF genes (HSF1 to -3) and the respective gene products (HSFs) have been characterized. We report the cloning and characterization of human HSF4 (hHSF4), a novel member of the hHSF family that shares properties with other members of the HSF family yet appears to be functionally distinct. hHSF4 lacks the carboxyl-terminal hydrophobic repeat which is shared among all vertebrate HSFs and has been suggested to be involved in the negative regulation of DNA binding activity. hHSF4 is preferentially expressed in the human heart, brain, skeletal muscle, and pancreas. Transient transfection of hHSF4 in HeLa cells, which do not express hHSF4, results in a constitutively active DNA binding trimer which, unlike other members of the HSF family, lacks the properties of a transcriptional activator. Constitutive overexpression of hHSF4 in HeLa cells results in reduced expression of the endogenous hsp70, hsp90, and hsp27 genes. hHSF4 represents a novel hHSF that exhibits tissue-specific expression and functions to repress the expression of genes encoding heat shock proteins and molecular chaperones.
PMCID: PMC231772  PMID: 8972228
14.  Mechanism for inhibition of influenza virus RNA polymerase activity by matrix protein. 
Journal of Virology  1996;70(1):241-247.
Influenza virus M1 protein has been shown to inhibit the transcription catalyzed by viral ribonucleoprotein complexes isolated from virions. Here, this inhibition mechanism was studied with the recombinant M1 protein purified from Escherichia coli expressing it from cDNA. RNA mobility shift assays indicated that both soluble and aggregate forms of the recombinant M1, which were separated by the glycerol density gradient, were bound to RNA. Once an M1-RNA complex was formed, free M1 was bound to the M1-RNA complex cooperatively rather than to free RNA. In addition, the recombinant M1 was capable of binding to preformed RNA-nucleocapsid protein complexes. The mechanism for inhibition of the viral RNA polymerase activity was analyzed by the in vitro RNA synthesis systems that depend on an exogenously added RNA template. These systems were more sensitive for evaluating the inhibition by M1 than the RNA synthesis system depending on an endogenous RNA template. The RNA synthesis inhibition was examined at four steps: cleavage of capped RNA; incorporation of the first nucleotide, GMP; limited elongation; and synthesis of full-size product. M1 inhibited RNA synthesis mainly at the early steps. The experiments with M1 mutant proteins containing amino acid deletions suggested that the M1 region between amino acid residues 91 and 111 was essential for anti-RNA synthesis activity, RNA binding, and oligomerization of M1 on RNA.
PMCID: PMC189810  PMID: 8523532
15.  A novel costimulatory factor for gamma interferon induction found in the livers of mice causes endotoxic shock. 
Infection and Immunity  1995;63(10):3966-3972.
Administration of monoclonal anti-CD3 antibody to mice treated with Propionibacterium acnes induced secretion of a high level of gamma interferon (IFN-gamma) into the circulation system, while it induced no significant release in untreated mice. In order to analyze this high-level induction of IFN-gamma in these bacterium-treated mice, we investigated the factors that might be involved. An activity that induces IFN-gamma in T cells was observed in the liver extracts of mice treated with P. acnes and subsequently challenged with lipopolysaccharide. Here, we purified an IFN-gamma-inducing factor from the liver extract to homogeneity and characterized it. Its molecular mass was 18 to 19 kDa, and its pI was 4.9. The amino acid sequence of the NH2-terminal portion was determined and shown to have no similarities to any protein in the EMBL, GenBank, and PIR data bases. The same molecule was also demonstrated in the serum factor that was previously reported to have an IFN-gamma-inducing activity and to have an apparent molecular mass of 75 kDa. Moreover, the activity of this serum factor was recovered in the fraction containing the 18- to 19-kDa protein under reducing conditions and was shown to have the same NH2-terminal amino acid sequence as that of the factor from the liver extract. In addition to the ability to induce IFN-gamma, this protein augmented T-cell proliferation and NK activity in the spleen cells. Thus, several of its biological activities were apparently similar to those of interleukin-12. These results indicated that this novel protein, which exhibited marked costimulatory activity on IFN-gamma production in vitro, was elevated vivo in response to P. acnes treatment. This factor, probably released from the producing cells by lipopolysaccharide stimuli, may be involved in the high-level induction of IFN-gamma in the P. acnes-treated mice.
PMCID: PMC173557  PMID: 7558306
16.  Growth inhibition of Ureaplasma urealyticum by the proton pump inhibitor lansoprazole: direct attribution to inhibition by lansoprazole of urease activity and urea-induced ATP synthesis in U. urealyticum. 
Antimicrobial Agents and Chemotherapy  1995;39(10):2187-2192.
The proton pump inhibitors (PPIs) omeprazole and lansoprazole and the acid-activated analog of lansoprazole AG-2000, which potently inhibit the urease of Helicobacter pylori (K. Nagata, H. Satoh, T. Iwahi, T. Shimoyama, and T. Tamura, Antimicrob. Agents Chemother. 37:769-774, 1993), also inhibited the urease activities of cell-free extracts as well as intact cells of Ureaplasma urealyticum. The 50% inhibitory concentrations were between 1 and 25 microM. These compounds also inhibited the ATP synthesis induced by urea in ureaplasma cells. The 50% inhibitory concentrations for ATP synthesis were close to those for urease activity, but they were lower than those of urease inhibitors, such as acetohydroxamic acid, hydroxyurea, and thiourea. In addition, one of the metabolites of lansoprazole found in human urine, M-VI, also inhibited ureaplasmal urease activity and the ATP synthesis induced by urea at almost the same concentrations as those of lansoprazole. The inhibition of PPIs against ureaplasma urease was very similar to those against H. pylori urease, suggesting that the inhibitory mechanism against these ureases was due to the blockage of the SH residues on the cysteine of the enzyme. Omeprazole, lansoprazole, AG-2000, and M-VI inhibited the growth of U. urealyticum. Since ureaplasma urease is thought to be involved in the pathogenicity of this organism in the urogenital tract, PPIs and their analogs may be useful as chemotherapeutic agents against diseases caused by U. urealyticum.
PMCID: PMC162911  PMID: 8619564
17.  The DNA-binding properties of two heat shock factors, HSF1 and HSF3, are induced in the avian erythroblast cell line HD6. 
Molecular and Cellular Biology  1995;15(10):5268-5278.
Avian cells express three heat shock transcription factor (HSF) genes corresponding to a novel factor, HSF3, and homologs of mouse and human HSF1 and HSF2. Analysis of the biochemical and cell biological properties of these HSFs reveals that HSF3 has properties in common with both HSF1 and HSF2 and yet has features which are distinct from both. HSF3 is constitutively expressed in the erythroblast cell line HD6, the lymphoblast cell line MSB, and embryo fibroblasts, and yet its DNA-binding activity is induced only upon exposure of HD6 cells to heat shock. Acquisition of HSF3 DNA-binding activity in HD6 cells is accompanied by oligomerization from a non-DNA-binding dimer to a DNA-binding trimer, whereas the effect of heat shock on HSF1 is oligomerization of an inert monomer to a DNA-binding trimer. Induction of HSF3 DNA-binding activity is delayed compared with that of HSF1. As occurs for HSF1, heat shock leads to the translocation of HSF3 to the nucleus. HSF exhibits the properties of a transcriptional activator, as judged from the stimulatory activity of transiently overexpressed HSF3 measured by using a heat shock element-containing reporter construct and as independently assayed by the activity of a chimeric GAL4-HSF3 protein on a GAL4 reporter construct. These results reveal that HSF3 is negatively regulated in avian cells and acquires DNA-binding activity in certain cells upon heat shock.
PMCID: PMC230774  PMID: 7565675
18.  Actinobacillus actinomycetemcomitans Y4 capsular-polysaccharide-like polysaccharide promotes osteoclast-like cell formation by interleukin-1 alpha production in mouse marrow cultures. 
Infection and Immunity  1995;63(5):1893-1898.
The mechanism of osteoclast-like cell formation induced by periodontopathic bacterium Actinobacillus actinomycetemcomitans Y4 (serotype b) capsular-polysaccharide-like polysaccharide (capsular-like polysaccharide) was examined in a mouse bone marrow culture system. When mouse bone marrow cells were cultured with A. actinomycetemcomitans Y4 capsular-like polysaccharide for 9 days, many multinucleated cells were formed. The multinucleated cells showed several characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRACP) and the ability to resorb the calcified dentine. In this study, we examined the effects of antisera to interleukins on the formation of osteoclast-like cells induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide. Monospecific anti-mouse recombinant interleukin-1 alpha (rIL-1 alpha) serum completely inhibited the formation of osteoclast-like cells in the presence of A. actinomycetemcomitans Y4 capsular-like polysaccharide. However, anti-mouse rIL-1 beta and anti-mouse rIL-6 sera showed no effect on osteoclast-like cell formation. IL-1 receptor antagonist significantly inhibited the osteoclast-like cell formation mediated by A. actinomycetemcomitans Y4 capsular-like polysaccharide in mouse marrow cultures. The bioactive IL-1 was detected in the culture media of mouse bone marrow cells stimulated with A. actinomycetemcomitans Y4 capsular-like polysaccharide. These results indicate that IL-1 alpha is involved in the mechanism of the formation of osteoclast-like cells induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide. We sought to determine whether osteoclast-like cell formation induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide could be modulated by the protein kinase inhibitors H8 and HA1004. The formation of osteoclast-like cells was suppressed by H8 and HA1004. These findings suggest that the signals by protein kinases may regulate osteoclast-like cell formation induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide. Furthermore, a correlation between IL-1 alpha and prostaglandin E2 in the osteoclast recruitment induced by A. actinomycetemcomitans Y4 capsular-like polysaccharide is discussed.
PMCID: PMC173240  PMID: 7729899
19.  Inhibitory action of lansoprazole and its analogs against Helicobacter pylori: inhibition of growth is not related to inhibition of urease. 
The proton pump inhibitors omeprazole and lansoprazole and its acid-activated derivative AG-2000, which are potent and specific inhibitors of urease of Helicobacter pylori (K. Nagata, H. Satoh, T. Iwahi, T. Shimoyama, and T. Tamura, Antimicrob. Agents Chemother. 37:769-774, 1993), inhibited the growth of H. pylori. The growth was inhibited not only in urease-positive clinical isolates but also in their urease-negative derivatives which had no urease polypeptides. AG-1789, a derivative of lansoprazole with no inhibitory activity against H. pylori urease, also inhibited the growth of both strains even more strongly than the urease inhibitors lansoprazole and AG-2000. Furthermore, the antibacterial activity of omeprazole and lansoprazole was not affected by glutathione or dithiothreitol, which completely abolished the inhibitory activity of lansoprazole against H. pylori urease. These results indicated that the inhibitory action of these compounds against the growth of H. pylori was independent from the inhibitory action against urease.
PMCID: PMC162584  PMID: 7726537
20.  Coexpression of the collagen-binding stress protein HSP47 gene and the alpha 1(I) and alpha 1(III) collagen genes in carbon tetrachloride-induced rat liver fibrosis. 
Journal of Clinical Investigation  1994;94(6):2481-2488.
HSP47 is a collagen-binding stress protein and is assumed to act as a collagen-specific molecular chaperone during the biosynthesis and secretion of procollagen in the living cell. The synthesis of HSP47 has been reported to correlate with that of collagen in several cell lines. We examined the expression of HSP47 mRNA during the progression of carbon tetrachloride (CCl4)-induced liver fibrosis in rats. Northern blot analysis revealed that the expression of HSP47 mRNA was markedly induced during the progression of fibrosis in parallel with alpha 1(I) and alpha 1(III) collagen mRNAs. By in situ hybridization, the distribution of HSP47 transcripts was similar to that of alpha 1(I) collagen and was observed only in cells lining collagen fibrils. These collagen-positive cells were confirmed to be Ito cells by immunohistochemistry for desmin. The absence of high levels of HSP47 mRNA in the liver of rats treated with only a single administration of CCl4 indicated that the induction of HSP47 mRNA was not due to the direct effect of CCl4 as a stressor, but was due to the progression of liver fibrosis. The function of HSP47 in liver fibrosis will also be discussed.
PMCID: PMC330081  PMID: 7989606
21.  Regulation of influenza virus RNA polymerase activity by cellular and viral factors. 
Nucleic Acids Research  1994;22(23):5047-5053.
An in vitro RNA synthesis system mimicking replication of genomic influenza virus RNA was developed with nuclear extracts prepared from influenza virus-infected HeLa cells using exogenously added RNA templates. The RNA synthesizing activity was divided into two complementing fractions, i.e. the ribonucleoprotein (RNP) complexes and the fraction free of RNP, which could be replaced with RNP cores isolated from virions and nuclear extracts from uninfected cells, respectively. When nuclear extracts from uninfected cells were fractionated by phosphocellulose column chromatography, the stimulatory activity for RNA synthesis was further separated into two distinct fractions. One of them, tentatively designated RAF (RNA polymerase activating factor), stimulated RNA synthesis with either RNP cores or RNA polymerase and nucleocapsid protein purified from RNP cores as the enzyme source. In contrast, the other, designated PRF (polymerase regulating factor), functioned as an activator only when RNP cores were used as the enzyme source. Biochemical analyses revealed that PRF facilitates dissociation of RNA polymerase from RNP cores. Of interest is that virus-coded non-structural protein 1 (NS1), which has been thought to be involved in regulation of replication, counteracted PRF function. Roles of cellular factors and viral proteins, NS1 in particular, are discussed in terms of regulation of influenza virus RNA genome replication.
PMCID: PMC523776  PMID: 7800498
22.  Characterization and expression of hepatic sulfotransferase involved in the metabolism of N-substituted aryl compounds. 
Environmental Health Perspectives  1994;102(Suppl 6):99-103.
An aryl sulfotransferase, whose cDNA was isolated from the rat liver library, was found to catalyze bioactivation of minoxidil through N-O-sulfation and N-sulfation of a carcinogenic heterocyclic amine, IQ, by expression in COS-1 cells. cDNA of a human ortholog also was isolated and characterized as a major minoxidil-activating enzyme in human liver. Another group of aryl sulfotransferases catalyzing O-sulfation of carcinogenic N-hydroxyarylamines was separated from livers of rats and humans. These sulfotransferases have been shown to possess similar functional properties and also to relate immunochemically with each other. Current understanding on the primary structure of these sulfotransferases also is discussed.
PMCID: PMC1566861  PMID: 7889867
23.  Alternative 5' splice site selection induced by heat shock. 
Molecular and Cellular Biology  1994;14(1):567-575.
The mouse HSP47 gene consists of six exons separated by five introns. Three HSP47 cDNAs differing only in their 5' noncoding regions have been reported. One of these alternatively spliced mRNAs was detected only after heat shock, which caused an alternative 5' splice donor site selection. Other stress inducers, including an amino acid analog and sodium arsenite, had no effect on the alternative splicing. The alternatively spliced mRNA, which was 169 nucleotides longer in the 5' noncoding region compared to mRNA transcribed in non-heat shock conditions, was efficiently translated under heat shock conditions. This novel finding that alternative splicing is caused by artificial treatment like heat shock will provide a useful in vivo model for understanding the exon-intron recognition mechanism as well as heat shock-induced alterations in gene expression.
PMCID: PMC358406  PMID: 8264624
24.  Differential effect of pharmacological autonomic blockade on some electrophysiological properties of the human ventricle and atrium. 
British Heart Journal  1994;71(1):34-37.
OBJECTIVE--This study investigated the dominance of each limb of the autonomic nervous system and tested sympathetic-vagal interactions in the human ventricle and atrium after administration of propranolol and atropine. PATIENTS AND METHODS--The 90% monophasic action potential duration (MAPD90) and the effective refractory period (ERP) at the right ventricular apex (RV) and the right lateral atrium (RA) were measured in 14 patients. The MAPD90 was measured during constant RV and RA pacing (cycle length 600 ms) and the ERP was measured at a driven cycle length of 600 ms. Electrophysiological variables were measured during a control period, after propranolol (0.15 mg/kg loading dose followed by 0.1 mg/min infusion), and after autonomic blockade (atropine 0.04 mg/kg). RESULTS--Both RV MAPD90 and RV ERP increased after propranolol (RV MAPD90 from 268 (26) ms to 275 (26) ms, p < 0.005; RV ERP from 252 (25) ms to 258 (26) ms, p < 0.0005) and then decreased to below the control values after autonomic blockade (RV MAPD90 256 (24) ms; RV ERP 239 (25) ms, p < 0.0005 v propranolol, p < 0.0005 v control). In contrast, both RA MAPD90 and RA ERP increased after propranolol (RA MAPD90 from 242 (19) ms to 260 (19) ms; RA ERP from 216 (21) ms to 230 (18) ms, p < 0.0005), and then increased slightly more after autonomic blockade (RA MAPD90 265 (16) ms, p = 0.09; RA ERP 235 (16) ms, p = 0.07), thus remaining above control values (p < 0.0005). CONCLUSIONS--The results indicate (a) that in the human ventricle vagal stimulation and sympathetic beta stimulation are antagonistic and that direct vagal stimulation predominates over beta stimulation, with sympathetic-vagal interaction being minimal and (b) that in the human atrium vagal stimulation and beta stimulation are synergistic and beta stimulation predominates over vagal stimulation, with direct vagal stimulation having a minimal effect.
PMCID: PMC483606  PMID: 8297691
25.  Cell-type-specific function of the C-type natriuretic peptide gene promoter in rat anterior pituitary-derived cultured cell lines. 
Molecular and Cellular Biology  1993;13(7):4077-4086.
The promoter function of the human C-type natriuretic peptide (CNP) gene in various cultured cells was examined by transient transfection assays. The CNP promoter functioned very effectively in GH3 cells, which originated from the growth hormone-producing tumor of the rat anterior pituitary and somatomammotroph phenotype, but functioned much less effectively in GH1 cells, another type of rat pituitary-derived cell with a somatotroph phenotype, and rat primary cardiocytes. The CNP promoter did not function at all in other cells, including AtT20 cells of murine pituitary corticotroph origin. Functional analyses of the deleted promoters with various 5' deletion breakpoints revealed the existence of at least two negative and one positive regulatory regions. Within the positive regulatory region (positions -54 to -19), which conferred 90% of the promoter activity in GH3 cells, two equipotent GC-rich cis elements (positions -49 to -45 and -40 to -35) were identified. Both sites shared half of the promoter activity and binding properties to the nuclear protein in GH3 cells. Rat anterior pituitary tissue contained the binding protein of the identified cis element, which was identical or similar to that of GH3 cells. With Southwestern (DNA-protein) analysis, a 70-kDa specific binding protein distinct from known factors such as SP-1, AP-2, and Pit-1 was identified in the nuclear extract of GH3 cells.
PMCID: PMC359957  PMID: 8321215

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